Human 2-19 protein homologue, z219a

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for z219a, a novel member of the human 2-19 protein family. The polypeptides, and polynucleotides encoding them, may be used for detecting human chromosomal abnormalities. The present invention also includes antibodies to the z219a polypeptides.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to Provisional Application No.60/061,712, filed on Oct. 6, 1997. Under 35 U.S.C. §119(e)(1), thisapplication claims benefit of said Provisional Application.Additionally, this continuation application claims benefit ofapplication Ser. No. 09/167,513 filed on Oct. 6, 1998, under 35 U.S.C.35 §20.

BACKGROUND OF THE INVENTION

[0002] Proliferation and differentiation of cells of multicellularorganisms are controlled by hormones and polypeptide growth factors.These diffusable molecules allow cells to communicate with each otherand act in concert to regulate cells and form organs, and to repair andregenerate damaged tissue. Examples of hormones and growth factorsinclude the steroid hormones (e.g. estrogen, testosterone), parathyroidhormone, follicle stimulating hormone, the interleukins, plateletderived growth factor (PDGF), epidermal growth factor (EGF),granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

[0003] Hormones and growth factors influence cellular metabolism bybinding to proteins. These proteins may be integral membrane proteinsthat are linked to signaling pathways within the cell, such as secondmessenger systems. Other classes of proteins that hormones and growthfactors influence are soluble molecules, such as the transcriptionfactors.

[0004] There is a continuing need to discover new hormones, growthfactors and the like. The in vivo activities of these cytokinesillustrates the enormous clinical potential of, and need for, othercytokines, cytokine agonists, and cytokine antagonists. The presentinvention addresses this need by providing such polypeptides for theseand other uses that should be apparent to those skilled in the art fromthe teachings herein.

SUMMARY OF THE INVENTION

[0005] The present invention addresses this need by providing a novelpolypeptide and related compositions and methods.

[0006] Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a polypeptide comprising a sequence of aminoacid residues that is at least 90% identical to an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 26 (Tyr), to amino acidnumber 235 (Ser); and (b) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 1 (Met), to amino acid number 235 (Ser).Within one embodiment, the isolated polynucleotide molecule is selectedfrom the group consisting of: (a) polynucleotide molecules comprising anucleotide sequence as shown in SEQ ID NO:1 from nucleotide 194 tonucleotide 823; (b) polynucleotide molecules comprising a nucleotidesequence as shown in SEQ ID NO:1 from nucleotide 119 to nucleotide 823;and (c) polynucleotide molecules complementary to (a) or (b). Withinanother embodiment, the isolated polynucleotide disclosed abovecomprises nucleotide 1 to nucleotide 705 of SEQ ID NO:8. Within anotherembodiment, the isolated polynucleotide disclosed above consists of asequence of amino acid residues that is at least 90% identical to anamino acid sequence as shown in SEQ ID NO:2 from amino acid number 26(Tyr), to amino acid number 235 (Ser). Within another embodiment, theisolated polynucleotide disclosed above consists of a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid number 26 (Tyr),to amino acid number 235 (Ser). Within another embodiment, the isolatedpolynucleotide disclosed above encodes a polypeptide that containsmotifs 1, 2, 3, 4 and 5 spaced apart from N-terminus to C-terminus in aconfiguration M1-{25-26}-M2-{15}-M3-{11}-M4-{34-36}-M5.

[0007] Within a second aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a z219a polypeptide thatis at least 90% identical to an amino acid sequence as shown in SEQ IDNO:2 from amino acid number 26 (Tyr), to amino acid number 235 (Ser);and a transcription terminator, wherein the promoter is operably linkedto the DNA segment, and the DNA segment is operably linked to thetranscription terminator. Within one embodiment, the expression vectordisclosed above, further comprises a secretory signal sequence operablylinked to the DNA segment.

[0008] Within a third aspect, the present invention provides, a culturedcell into which has been introduced an expression vector as disclosedabove, wherein the cell expresses the polypeptide encoded by the DNAsegment.

[0009] Within a fourth aspect, the present invention provides a DNAconstruct encoding a fusion protein, the DNA construct comprising: afirst DNA segment encoding a polypeptide that is at least 90% identicalto a sequence of amino acid residues 1 (Met) through 25 (Gly) of SEQ IDNO:2; and second DNA segment encoding an additional polypeptide, whereinthe first and second DNA segments are connected in-frame; and encode thefusion protein.

[0010] Within another aspect, the present invention provides an isolatedpolypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) polypeptide molecules comprising an amino acidsequence as shown in SEQ ID NO:2 from amino acid number 26 (Tyr) toamino acid number 235 (Ser) of SEQ ID NO:2; (b) polypeptide moleculescomprising an amino acid sequence as shown in SEQ ID NO:2 from aminoacid residue number 1 (Met) to amino acid residue number 235 (Ser).Within one embodiment, the isolated polypeptide disclosed above consistsof a sequence of amino acid residues that is at least 90% identical toan amino acid sequence as shown in SEQ ID NO:2 from amino acid number 26(Tyr) to amino acid number 235 (Ser). Within another embodiment, theisolated polypeptide disclosed above is as shown in SEQ ID NO:2 fromamino acid number 26 (Tyr) to amino acid number 235 (Ser). Withinanother embodiment, the isolated polypeptide disclosed above encodesmotifs 1, 2, 3, 4 and 5 spaced apart from N-terminus to C-terminus in aconfiguration M1-{25-26}-M2-{15}-M3-{11}-M4-{34-36}-M5.

[0011] Within another aspect, the present invention provides a method ofproducing a z219a polypeptide comprising: culturing a cell as disclosedabove; and isolating the z219a polypeptide produced by the cell.

[0012] Within another aspect, the present invention provides a method ofproducing an antibody to z219a polypeptide comprising: inoculating ananimal with a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 9 to 210 amino acids, wherein the polypeptideis at least 90% identical to a contiguous sequence of amino acids in SEQID NO:2 from amino acid number 26 (Tyr) to amino acid number 235 (Ser);(b) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2from amino acid number 26 (Tyr) to amino acid number 235 (Ser); (c) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 59 (Arg) to amino acid number 133 (Asp); (d) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 135 (Ser) to amino acid number 212 (Ala); (e) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid 215 (Asn) to amino acid number 231 (Pro); and wherein thepolypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal.

[0013] Within another aspect, the present invention provides an antibodyproduced by the method disclosed above, which specifically binds to az219a polypeptide. Within one embodiment, the antibody disclosed aboveis a monoclonal antibody.

[0014] Within another aspect, the present invention provides an antibodywhich specifically binds to a polypeptide disclosed above.

[0015] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an antagonist of z219aprotein activity, comprising: transfecting a z219a-responsive cell, witha reporter gene construct that is responsive to a z219a-stimulatedcellular pathway; and producing a z219a polypeptide by the methoddisclosed above; and adding the z219a polypeptide to the cell, in thepresence and absence of a test sample; and comparing levels of responseto the z219a polypeptide, in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison, the presence of the antagonist of z219a activity in the testsample.

[0016] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an agonist of z219a proteinactivity, comprising: transfecting a z219a-responsive cell, with areporter gene construct that is responsive to a z219a-stimulatedcellular pathway; and adding a test sample; and comparing levels ofresponse in the presence and absence of the test sample, by a biologicalor biochemical assay; and determining from the comparison, the presenceof the agonist of z219a activity in the test sample.

[0017] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The FIGURE illustrates a multiple alignment of murine EF-7protein (MMU72677_(—)1), human 2-19 protein (219_HUMAN), human D87120(D87120_(—)1_|), and z219a (z219a.pep).

DETAILED DESCRIPTION OF THE INVENTION

[0019] Prior to setting forth the invention in detail, it is helpful tothe understanding thereof to define the

[0020] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0021] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0022] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0023] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0024] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0025] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.

[0026] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0027] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, andthe like. Expression vectors are generally derived from plasmid or viralDNA, or may contain elements of both.

[0028] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0029] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0030] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0031] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0032] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0033] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

[0034] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0035] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0036] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0037] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

[0038] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide” ) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0039] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0040] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0041] Teachings of all references cited herein are in their entiretyincorporated by reference.

[0042] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a polypeptide having homology to human2-19 protein and human cancellous bone protein D87120. Analysis of thetissue distribution of the mRNA corresponding to this novel DNA showedthat expression was highest in pancreas, followed by moderately strongexpression levels in small intestine, trachea and prostate. Relativelyless intense signals were present in placenta, kidney, testis, stomach,and thyroid. A weaker signal was present in lung. The polypeptide hasbeen designated z219a.

[0043] Novel polypeptides, such as z219a, are initially identified byquerying an EST database. An identified EST can be extended to fulllength by conventional techniques, as is described below and inExample 1. Generally, one or a combination of several techniques couldbe used to obtain the full length sequence of the z219apolypeptide-encoding polynucleotide. First, if one or more additionalESTs are identified that are contigs to the identified EST sequence,cDNA clones corresponding to such ESTs can be sequenced and splicedtogether with the original sequence to form the full length sequence. Ifa small portion of the full length sequence is absent, 5′ RACE reactionscan be done, and the resulting fragments can be sequenced and splicedtogether with the original sequence to form the full length sequence.Also, one or more cDNA libraries can be probed with all or a portion ofan EST sequence to identify a putative full-length clone.

[0044] The novel z219a polypeptides of the present invention wereinitially identified by querying an EST database for proteins homologousto proteins having a secretory signal sequence. These proteins arecharacterized by an upstream methionine start site and a hydrophobicregion of approximately 13 amino acids. Polypeptides meeting thosesearch criteria were compared to an EST database to identify secretedproteins having homology to known ligands. A single EST sequence wasdiscovered and predicted to code for part of a secreted protein. Acontig to this initial EST was found and its corresponding full-lengthcDNA was sequenced. The novel polypeptide encoded by the full lengthcDNA showed homology with the human 2-19 (Genbank accession No. X55448)and murine EF-7 protein family (Fu, X. and Kamps, M. P., Mol. Cell.Biol., 17;l503-1511, 1997).

[0045] The full sequence of the z219a polypeptide was obtained from asingle clone believed to contain it, wherein the clone was obtained froma gastrointestinal tissue library. Other libraries that might also besearched for such sequences include pancreas, trachea, salivary gland,prostate, bone marrow, testis, mammary gland and the like.

[0046] The nucleotide sequence of full-length z219a is described in SEQID NO. 1, and its deduced amino acid sequence is described in SEQ ID NO.2. The multiple alignment (FIGURE) revealed that z219a is a member of afamily of proteins that are characterized by their signal sequence,predicted small size (15-40 kD), tissue-specific expression, certainnovel motifs disclosed herein, glycosylation sites, and lack of longhydrophobic segments, suggesting a small secreted molecule withpotential as a new class of secreted cytokine-like molecules.

[0047] Analysis of the DNA encoding z219a polypeptide (SEQ ID NO:1)revealed an open reading frame encoding 235 amino acids (SEQ ID NO:2)comprising a predicted signal peptide of 25 amino acid residues (residue1 (Met) to residue 25 (Gly) of SEQ ID NO:2), and a mature polypeptide of210 amino acids (residue 26 (Tyr) to residue 235 (Ser) of SEQ ID NO:2).Multiple alignment of z219a with other members of the human 2-19 proteinfamily revealed the following 3 regions of conserved amino acids (seeFigure):

[0048] 1) The first region, referred to hereinafter as “block 1,”corresponds to amino acid residues 59 (Arg) to amino acid residue 133(Asp) of SEQ ID NO:2. Within block 1 there is one conserved motifreferred to hereinafter as “motif 1” (SEQ ID NO:3; corresponding toamino acids 127 to 129 of SEQ ID NO:2).

[0049] 2) The second region, referred to hereinafter as “block 2,”corresponds to amino acid residues 135 (Ser) to amino acid residue 212(Ala) of SEQ ID NO:2. Within block 2 there are three conserved motifsreferred to hereinafter as “motif 2” (SEQ ID NO:4; corresponding toamino acids 156 to 158 of SEQ ID NO:2), “motif 3” (SEQ ID NO:5; aminoacids 174 to 176 of SEQ ID NO:2), and “motif 4” (SEQ ID NO:6; aminoacids 188 to 190 of SEQ ID NO:2). Blocks 1 and 2 are separated by asingle Asn residue present in z219a but not in other human 2-19 proteinfamily members.

[0050] 3) The third region, referred to hereinafter as “block 3,”corresponds to amino acid residues 215 (Asn) to amino acid residue 231(Pro) of SEQ ID NO:2. Within block 3 there is one conserved motifreferred to hereinafter as “motif 5” (SEQ ID NO:7; corresponding toamino acids 227 to 229 of SEQ ID NO:2).

[0051] Motifs 1 through 5 are spaced apart from N-terminus to C-terminusin a configuration represented by the following:

[0052] M1-{25-26}-M2-{15}-M3-{11}-M4-{34-36}-M5,

[0053] where

[0054] M# denotes the specific motif disclosed above and

[0055] {#} denotes the number of amino acids between the motifs.

[0056] The presence of transmembrane regions, and conserved and lowvariance motifs generally correlates with or defines importantstructural regions in proteins. Regions of low variance (e.g.,hydrophobic clusters) are generally present in regions of structuralimportance (Sheppard, P. et al., supra.). Such regions of low varianceoften contain rare or infrequent amino acids, such as Tryptophan. Theregions flanking and between such conserved and low variance motifs maybe more variable, but are often functionally significant because theymay relate to or define important structures and activities such asbinding domains, biological and enzymatic activity, signal transduction,cell-cell interaction, tissue localization domains and the like.

[0057] Moreover, z219a polypeptide has two predicted glycosylation sitesfor z219a located at amino acids 120 (Asn) and 208 (Asn) in SEQ ID NO:2.The predicted glycosylation site at amino acid 208 is conservedthroughout the family. The corresponding polynucleotides encoding thez219a polypeptide regions, domains, motifs, residues and sequencesdescribed above are as shown in SEQ ID NO:1.

[0058] The highly conserved amino acids in motifs 1 through 5 of z219acan be used as a tool to identify new family members. For instance,reverse transcription-polymerase chain reaction (RT-PCR) can be used toamplify sequences encoding the conserved motifs from RNA obtained from avariety of tissue sources or cell lines. In particular, highlydegenerate primers designed from the z219a sequences are useful for thispurpose.

[0059] The present invention also provides polynucleotide molecules,including DNA and RNA molecules, that encode the z219a polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:8is a degenerate DNA sequence that encompasses all DNAs that encode thez219a polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:8 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, z219apolypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 705 of SEQ ID NO:8 and their RNA equivalents are contemplatedby the present invention. Table 1 sets forth the one-letter codes usedwithin SEQ ID NO:8 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C. TABLE 1Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T TA A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T WA|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T NA|C|G|T N A|C|G|T

[0060] The degenerate codons used in SEQ ID NO:8, encompassing allpossible codons for a given amino acid, are set forth in Table 2 below.TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

[0061] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY) . Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0062] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:8 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0063] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Suitable stringent hybridization conditions areequivalent to about a 5 h to overnight incubation at about 42° C. in asolution comprising: about 40-50% formamide, up to about 5×SSC, about5×Denhardt's solution, up to about 10% dextran sulfate, and about 10-20μg/ml denatured commercially-available carrier DNA; hybridization isthen followed by washing filters in up to about 2×SSC. For example, asuitable wash stringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at55° C. to 65° C. Stringent hybridization and wash conditions depend onthe length of the probe, reflected in the T_(m), hybridization and washsolutions used, and are routinely determined empirically by one of skillin the art.

[0064] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of z219a RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include pancreas, small intestine or prostatealthough DNA can also be prepared using RNA from other tissues or celllines or isolated as genomic DNA. Total RNA can be prepared usingguanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412,1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA usingknown methods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding z219a polypeptides are then identified andisolated by, for example, hybridization or PCR.

[0065] A full-length clone encoding z219a can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to z219a, receptor fragments, orother specific binding partners.

[0066] The polynucleotides of the present invention can also besynthesized using DNA synthesis machines. Currently the method of choiceis the phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

[0067] One method for building a synthetic gene requires the initialproduction of a set of overlapping, complementary oligonucleotides, eachof which is between 20 to 60 nucleotides long. Each internal section ofthe gene has complementary 3′ and 5′ terminal extensions designed tobase pair precisely with an adjacent section. Thus, after the gene isassembled, process is completed by sealing the nicks along the backbonesof the two strands with T4 DNA ligase. In addition to the protein codingsequence, synthetic genes can be designed with terminal sequences thatfacilitate insertion into a restriction endonuclease site of a cloningvector.

[0068] An alternative way to prepare a full-length gene is to synthesizea specified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions areannealed, large gaps still remain, but the short base-paired regions areboth long enough and stable enough to hold the structure together. Thegaps are filled and the DNA duplex is completed via enzymatic DNAsynthesis by E. coli DNA polymerase I. After the enzymatic synthesis iscompleted, the nicks are sealed. Double-stranded constructs aresequentially linked to one another to form the entire gene sequencewhich is verified by DNA sequence analysis. See Glick and Pasternak,Molecular Biotechnology, Principles & Applications of Recombinant DNA,(ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem.53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7,1990.

[0069] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs) . These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are z219a polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human z219a can be cloned usinginformation and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses z219a as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A z219a-encoding cDNA can then be isolatedby a variety of methods, such as by probing with a complete or partialhuman cDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the representative human z219a sequence disclosed herein.Within an additional method, the cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to z219a polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

[0070] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human z219a andthat allelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the z219a polypeptide are included within the scope of thepresent invention, as are polypeptides encoded by such cDNAs and mRNAs.Allelic variants and splice variants of these sequences can be cloned byprobing cDNA or genomic libraries from different individuals or tissuesaccording to standard procedures known in the art.

[0071] The present invention also provides isolated z219a polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO:2 andtheir orthologs. The term “substantially similar” is used herein todenote polypeptides having 50%, preferably 60%, more preferably at least80%, sequence identity to the sequences shown in SEQ ID NO:2 or theirorthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2 orits orthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blosum 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 3 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0072] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0073] Variant z219a polypeptides or substantially homologous z219apolypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), maltose binding protein (Kellerman and Ferenci,Methods Enzymol. 90:459-463, 1982; Guan et al., Gene 67:21-30, 1987),thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, orother antigenic epitope or binding domain. See, in general Ford et al.,Protein Expression and Purification 2: 95-107, 1991. DNAs encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.). Thepresent invention thus includes polypeptides of from about 195 to about250 amino acid residues that comprise a sequence that is at least 80%,preferably at least 90%, and more preferably 95% or more identical tothe corresponding region of SEQ ID NO:2. Polypeptides comprisingaffinity tags can further comprise a proteolytic cleavage site betweenthe z219a polypeptide and the affinity tag. Preferred such sites includethrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0074] The present invention further provides a variety of otherpolypeptide fusions and related multimeric proteins comprising one ormore polypeptide fusions. For example, a z219a polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains. Immunoglobulinz219a polypeptide fusions can be expressed in genetically engineeredcells to produce a variety of multimeric z219a analogs. Auxiliarydomains can be fused to z219a polypeptides to target them to specificcells, tissues, or macromolecules (e.g., collagen). For example, a z219apolypeptide or protein could be targeted to a predetermined cell type byfusing a z219a polypeptide to a ligand that specifically binds to areceptor on the surface of the target cell. In this way, polypeptidesand proteins can be targeted for therapeutic or diagnostic purposes. Az219a polypeptide can be fused to two or more moieties, such as anaffinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9,1996.

[0075] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0076] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for z219a amino acidresidues.

[0077] Essential amino acids in the z219a polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological or biochemicalactivity (e.g., in situ localization or expression of z219a; secretionfollowed by detection by antibodies; or activity measured by a signaltransduction type assay) as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor or other biological interaction can also be determinedby physical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related familymembers.

[0078] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0079] Variants of the disclosed z219a DNA and polypeptide sequences canbe generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0080] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., secreted and detectedby antibodies; or measured by a signal transduction type assay) can berecovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

[0081] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially homologous to SEQ ID NO:2 or allelic variants thereof andretain the properties of the wild-type protein. For example, using themethods described above, one could identify a receptor binding domain onz219a; an extracellular ligand-binding domain of a receptor for z219a;heterodimeric and homodimeric binding domains; other functional orstructural domains; affinity tags; or other domains important forprotein-protein interactions or signal transduction. Such polypeptidesmay also include additional polypeptide segments as generally disclosedabove.

[0082] For any z219a polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above.

[0083] The z219a polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Bioloqy, John Wiley and Sons, Inc., NY, 1987.

[0084] In general, a DNA sequence encoding a z219a polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator, within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0085] To direct a z219a polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the z219a polypeptide, ormay be derived from another secreted protein (e.g., t-PA) or synthesizedde novo. The secretory signal sequence is operably linked to the z219aDNA sequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0086] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from residue 1 (Met) toresidue 25 (Gly) of SEQ ID NO:2 is be operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

[0087] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (A. Miller and G. Rosman, BioTechniques7:980-90, 1989; Q. Wang and M. Finer, Nature Med. 2:714-16, 1996). Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable culturedmammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293(ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) andChinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter.

[0088] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0089] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987 and WIPO publication WO 94/06463. Transformation of insect cellsand production of foreign polypeptides therein is disclosed by Guarinoet al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No. 4,775,624;and WIPO publication WO 94/06463. The use of Agrobacterium rhizogenes asa vector for expressing genes in plant cells has been reviewedby Sinkaret al., J. Biosci. (Bangalore) 11:47-58, 1987.

[0090] Fungal cells, including yeast cells, and particularly cells ofthe genus Saccharomyces, can also be used within the present invention,such as for producing z219a fragments or polypeptide fusions. Methodsfor Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See, King, L. A. and Possee, R. D., The Baculovirus Expression System: ALaboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et al.,Baculovirus Expression Vectors: A Laboratory Manual, New York, OxfordUniversity Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. A second method of making recombinant z219a baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system, which utilizes transfervectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville,Md.). This system utilizes a transfer vector, pFastBac1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding thez219a polypeptide into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins, M. S. and Possee, R.D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed z219a polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usinga technique known in the art, a transfer vector containing z219a istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses z219a is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0091] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF ₉₂₁™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification ofthe z2l9a polypeptide from the supernatant can be achieved using methodsdescribed herein.

[0092] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0093] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (τ) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0094] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0095] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a z219apolypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0096] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0097] Expressed recombinant z219a polypeptides (or chimeric z219apolypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for binding ligandor receptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

[0098] The polypeptides of the present invention can be isolated byexploitation of their structural and biological properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins, including those comprising polyhistidinetags. Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification” , M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

[0099] Moreover, using methods described in the art, polypeptidefusions, or hybrid z219a proteins, are constructed using regions ordomains of the inventive z219a in combination with those of other human2-19 family proteins (e.g. human 2-19, D87120, and murine EF-7), orheterologous proteins (Sambrook et al., ibid., Altschul et al., ibid.,Picard. D. Cur. Opin. Biology, 5:511-515, 1994, and references therein).These methods allow the determination of the biological importance oflarger domains or regions in a polypeptide of interest. Such hybrids mayalter reaction kinetics, binding, constrict or expand the substratespecificity, or alter tissue and cellular localization of a polypeptide,and can be applied to polypeptides of unknown structure.

[0100] Fusion polypeptides can be prepared by methods known to thoseskilled in the art by preparing each component of the fusion protein andchemically conjugating them. Alternatively, a polynucleotide encodingone or more components of the fusion protein in the proper reading framecan be generated using known techniques and expressed by the methodsdescribed herein. For example, part or all of a domain(s) conferring abiological function may be swapped between z219a of the presentinvention with the functionally equivalent domain(s) from another familymember, such as human 2-19 protein or D87120. Such domains include, butare not limited to, the secretory signal sequence, conserved motifs, andblocks 1, 2, and 3. Such fusion proteins would be expected to have abiological functional profile that is the same or similar topolypeptides of the present invention or other known 2-19 familyproteins (e.g. human 2-19, D87120, and murine EF-7) or heterologousproteins, depending on the fusion constructed. Moreover, such fusionproteins may exhibit other properties as disclosed herein.

[0101] Standard molecular biological and cloning techniques can be usedto swap the equivalent domains between the z219a polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., a z219a domain described herein, isoperably linked in frame to at least one other DNA segment encoding anadditional polypeptide (for instance an analogous domain or region human2-19 protein), and inserted into an appropriate expression vector, asdescribed herein. Generally DNA constructs are made such that theseveral DNA segments that encode the corresponding regions of apolypeptide are operably linked in frame to make a single construct thatencodes the entire fusion protein, or a functional portion thereof. Forexample, a DNA construct would encode from N-terminus to C-terminus afusion protein comprising a signal polypeptide followed by a maturepolypeptide; or a DNA construct would encode from N-terminus toC-terminus a fusion protein comprising a signal polypeptide followed byblock 1, followed by block2, followed by block 3. Such fusion proteinscan be expressed, isolated, and assayed for activity as describedherein.

[0102] Z219a polypeptides or fragments thereof may also be preparedthrough chemical synthesis. Z219a polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0103] In view of the tissue distribution observed for this z219apolypeptide, agonists (including the naturalligand/substrate/cofactor/etc.) and antagonists have enormous potentialin both in vitro and in vivo applications. Compounds identified as z219aagonists are useful for growth, proliferation or differentiation ofvarious cell types in vitro and treatment of diabetes, intestinalregeneration, gastric mucositis, mucosal regeneration, metabolicdisorders, prostate disorders in vivo. For example, agonist compoundsare useful as components of defined cell culture media, and may be usedalone or in combination with other cytokines and hormones to replaceserum that is commonly used in cell culture.

[0104] Within one embodiment, there is provided a method of identifyingagonists of z219a polypeptide, comprising providing cells responsive toa z219a polypeptide, culturing a first portion of the cells in thepresence of a test compound, culturing a second portion of the cells inthe absence of a test compound, and detecting an increase in a cellularresponse of the first portion of the cells as compared to the secondportion of the cells.

[0105] Within another embodiment, there is provided a method ofidentifying antagonists of z219a polypeptide, comprising providing cellsresponsive to a z219a polypeptide, culturing a first portion of thecells in the presence of z219a polypeptide, culturing a second portionof the cells in the presence of the z219a polypeptide and a testcompound, and detecting a decrease in a cellular response of the secondportion of the cells as compared to the first portion of the cells.

[0106] The activity of molecules of the present invention can bemeasured using a variety of assays that measure, for example, signaltransduction upon binding a receptor, mucosal secretion, antibodybinding, or ELISA. For example, z219a polypeptides can be labeled andtested for specific and saturating binding to specific cell lines orcells. After identification of positive cells to which z219a binds,activity can be tested for z219a-mediated activation of a signaltransduction pathway using methods known in the art. For instance,vector constructs containing a reporter (e.g. SRE-luciferase orSTAT-luciferase or the like) can be introduced into the positive celllines; such cell lines, when exposed to conditioned media containingsecreted z219a protein, will demonstrate z219a-mediated signaltransduction activity through activation of the measurable reporter.Such assays are well known in the art. Specific assays include, but arenot limited to bioassays measuring signal transduction.

[0107] In addition, z219a polypeptides of the present invention can beused to study pancreatic cell proliferation or differentiation. Suchmethods of the present invention generally comprise incubating a cells,β cells, δ cells, F cells and acinar cells in the presence and absenceof z219a polypeptide, monoclonal antibody, agonist or antagonist thereofand observing changes in islet cell proliferation or differentiation.

[0108] A further aspect of the invention provides a method for studyinginsulin. Such methods of the present invention comprise incubatingadipocytes in a culture medium comprising z219a polypeptide, monoclonalantibody, agonist or antagonist thereof ± insulin and observing changesin adipocyte protein secretion or differentiation.

[0109] The present invention also provides methods of studying mammaliancellular metabolism. Such methods of the present invention compriseincubating cells to be studied, for example, human vascular endothelialcells, ± z219a polypeptide, monoclonal antibody, agonist or antagonistthereof and observing changes in adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, or the like.

[0110] A high level of expression of z219a polypeptide was observed byNorthern blot in the trachea and by Dot blot in the salivary gland andtrachea. Consequently, another aspect of the present invention involvesthe detection of z219a polypeptides in the serum or tissue biopsy of apatient undergoing evaluation for salivary gland function ordysfunction. Such z219a polypeptides can be detected using immunoassaytechniques and antibodies capable of recognizing z219a polypeptideepitopes.

[0111] More specifically, the present invention contemplates methods fordetecting z219a polypeptide comprising:

[0112] exposing a solution possibly containing z219a polypeptide to anantibody attached to a solid support, wherein said antibody binds to afirst epitope of a z219a polypeptide;

[0113] washing said immobilized antibody-polypeptide to remove unboundcontaminants;

[0114] exposing the immobilized antibody-polypeptide to a secondantibody directed to a second epitope of a z219a polypeptide, whereinthe second antibody is associated with a detectable label; and

[0115] detecting the detectable label. Serum or biopsy z219a polypeptideconcentration (relative to normal serum or tissue concentration) may beindicative of dysfunction of the salivary gland.

[0116] Salivary gland dysfunction includes digestive dysfunction, woundhealing dysfunction, inadequate saliva production or composition,mucosal integrity breakdown, and failure of or diminished anti-microbialfunction. Detection of z219a polypeptide at relatively high levels inthe trachea may indicate that such polypeptides may serve as a marker oflung dysfunction. Moreover, z219a expression is detected in lung.Examples of conditions associated with salivary gland or lungdysfunction include salivary gland carcinoma, sarcoidosis, pneumocysticcarinii (particularly as associated with AIDS patients), emphysema,chronic bronchitis, cystic fibrosis, ARDS, SIDS or the like. Inaddition, z219a polypeptides are expressed in the prostate at a levelsimilar to trachea, as well as in the salivary gland. The prostate glandis androgen regulated and shares other properties with salivary glands.Consequently, dysfunction thereof, such as prostate adenocarcinoma orthe like, may also be detected using z219a polypeptides or z219aantibodies.

[0117] Also, the salivary glands synthesize and secrete a number ofproteins having diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins) anddigestion (e.g., amylase, lipase and proteases) and provideanti-microbial (e.g., proline-rich proteins, lysozyme, histatins andlactoperoxidase) and mucosal integrity maintenance (e.g., mucins)capabilities. In addition, saliva is a rich source of growth factorssynthesized by the salivary glands. For example, saliva is known tocontain epidermal growth factor (EGF), nerve growth factor (NGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), insulin, insulin-like growth factors I and II(IGF-I and IGF-II) and fibroblast growth factor (FGF). See, for example,Zelles et al., J. Dental. Res. 74(12): 1826-32, 1995. Synthesis ofgrowth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

[0118] Thus, z219a polypeptides, agonists or antagonists thereof may betherapeutically useful for aiding digestion. To verify the presence ofthis capability in z219a polypeptides, agonists or antagonists of thepresent invention, such z219a polypeptides, agonists or antagonists areevaluated with respect to their ability to break down starch accordingto procedures known in the art. If desired, z219a polypeptideperformance in this regard can be compared to digestive enzymes, such asamylase, lipase, proteases and the like. In addition, z219a polypeptidesor agonists or antagonists thereof may be evaluated in combination withone or more digestive enzymes to identify synergistic effects.

[0119] Also, z219a polypeptides, agonists or antagonists thereof may betherapeutically useful for promoting wound healing. To verify thepresence of this capability in z219a polypeptides, agonists orantagonists of the present invention, such z219a polypeptides, agonistsor antagonists are evaluated with respect to their ability to facilitatewound healing according to procedures known in the art. If desired,z219a polypeptide performance in this regard can be compared to growthfactors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II,fibroblast growth factor (FGF) and the like. In addition, z219apolypeptides or agonists or antagonists thereof may be evaluated incombination with one or more growth factors to identify synergisticeffects.

[0120] In addition, z219a polypeptides, agonists or antagonists thereofmay be therapeutically useful for anti-microbial applications. To verifythe presence of this capability in z219a polypeptides, agonists orantagonists of the present invention, such z219a polypeptides, agonistsor antagonists are evaluated with respect to their antimicrobialproperties according to procedures known in the art. See, for example,Barsum et al., Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica etal., J. Med. Vet. Mycol (England) 28 (4): 279-87, 1990; Mehentee et al.,J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage,Journal of Medical and Veterinary Mycology 24: 477-479, 1986 and thelike. If desired, z219a polypeptide performance in this regard can becompared to proteins known to be functional in this regard, such asproline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.In addition, z219a polypeptides or agonists or antagonists thereof maybe evaluated in combination with one or more antimicrobial agents toidentify synergistic effects.

[0121] The activity of molecules of the present invention can bemeasured using a variety of assays that measure stimulation ofgastrointestinal cell contractility, modulation of nutrient uptakeand/or secretion of digestive enzymes. Of particular interest arechanges in contractility of smooth muscle cells. For example, thecontractile response of segments of mammalian duodenum or othergastrointestinal smooth muscles tissue (Depoortere et al., J.Gastrointestinal Motility 1:150-159, 1989). An exemplary in vivo assayuses an ultrasonic micrometer to measure the dimensional changesradially between commissures and longitudinally to the plane of thevalve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390,1995).

[0122] Anti-microbial protective agents may be directly acting orindirectly acting. Such agents operating via membrane association orpore forming mechanisms of action directly attach to the offendingmicrobe. Anti-microbial agents can also act via an enzymatic mechanism,breaking down microbial protective substances or the cell wall/membranethereof. Anti-microbial agents, capable of inhibiting microorganismproliferation or action or of disrupting microorganism integrity byeither mechanism set forth above, are useful in methods for preventingcontamination in cell culture by microbes susceptible to thatanti-microbial activity. Such techniques involve culturing cells in thepresence of an effective amount of said z219a polypeptide or an agonistor antagonist thereof.

[0123] Also, z219a polypeptides or agonists thereof may be used as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection. Also, themicroorganism-adherence properties of z219a polypeptides or agoniststhereof can be studied under a variety of conditions in binding assaysand the like.

[0124] Moreover, z219a polypeptides, agonists or antagonists thereof maybe therapeutically useful for mucosal integrity maintenance. Tissueexpression of z219a is moderate to high in tissues involved in mucosalsecretion, such as small intestine, trachea and salivary gland. Toverify this presence of this capability in z219a polypeptides, agonistsor antagonists of the present invention, such z219a polypeptides,agonists or antagonists are evaluated with respect to their mucosalintegrity maintenance according to procedures known in the art. See, forexample, Zahm et al., Eur. Respir. J. 8: 381-6, 1995, which describesmethods for measuring viscoelastic properties and surface properties ofmucous as well as for evaluating mucous transport by cough and byciliary activity. If desired, z219a polypeptide performance in thisregard can be compared to mucins or the like. In addition, z219apolypeptides or agonists or antagonists thereof may be evaluated incombination with mucins to identify synergistic effects.

[0125] Proteins of the present invention are useful for example, intreating intestinal, prostate, and pancreatic disorders, and can bemeasured in vitro using cultured cells or in vivo by administeringmolecules of the present invention to the appropriate animal model. Forinstance, host cells expressing a z219a soluble receptor polypeptide canbe embedded in an alginate environment and injected (implanted) intorecipient animals. Alginate-poly-L-lysine microencapsulation,permselective membrane encapsulation and diffusion chambers are a meansto entrap transfected mammalian cells or primary mammalian cells. Thesetypes of non-immunogenic “encapsulations” permit the diffusion ofproteins and other macromolecules secreted or released by the capturedcells to the recipient animal. Most importantly, the capsules mask andshield the foreign, embedded cells from the recipient animal's immuneresponse. Such encapsulations can extend the life of the injected cellsfrom a few hours or days (naked cells) to several weeks (embeddedcells). Alginate threads provide a simple and quick means for generatingembedded cells.

[0126] The materials needed to generate the alginate threads are knownin the art. In an exemplary procedure, 3% alginate is prepared insterile H₂O, and sterile filtered. Just prior to preparation of alginatethreads, the alginate solution is again filtered. An approximately 50%cell suspension (containing about 5×10⁵ to about 5×10⁷ cells/ml) ismixed with the 3% alginate solution. One ml of the alginate/cellsuspension is extruded into a 100 mM sterile filtered CaCl₂ solutionover a time period of ˜15 min, forming a “thread” . The extruded threadis then transferred into a solution of 50 mM CaCl_(2,) and then into asolution of 25 mM CaCl₂. The thread is then rinsed with deionized waterbefore coating the thread by incubating in a 0.01% solution ofpoly-L-lysine. Finally, the thread is rinsed with Lactated Ringer'sSolution and drawn from solution into a syringe barrel (without needle).A large bore needle is then attached to the syringe, and the thread isintraperitoneally injected into a recipient in a minimal volume of theLactated Ringer's Solution.

[0127] An in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with many different promoters includingubiquitous, tissue specific, and regulatable promoters. Also, becauseadenoviruses are stable in the bloodstream, they can be administered byintravenous injection.

[0128] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene has been deleted from theviral vector, and the virus will not replicate unless the El gene isprovided by the host cell (the human 293 cell line is exemplary) . Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

[0129] Moreover, adenoviral vectors containing various deletions ofviral genes can be used in an attempt to reduce or eliminate immuneresponses to the vector. Such adenoviruses are E1 deleted, and inaddition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol.72:2022-2032, 1998; Raper, S. E. et al., Human Gene Therapy 9:671-679,1998). In addition, deletion of E2b is reported to reduce immuneresponses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover,by deleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses where all viral genes are deleted are particularlyadvantageous for insertion of large inserts of heterologous DNA. Forreview, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.

[0130] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

[0131] Compounds identified as z219a agonists are useful in vitro and invivo. For example, z219a and agonist compounds are useful as componentsof defined cell culture media, and may be used alone or in combinationwith other cytokines and hormones to replace serum that is commonly usedin cell culture. Thus, z219a polypeptides and z219a agonist polypeptidesare useful as a research reagent, such as for the expansion of culturedcells. As such, z219a polypeptides are added to tissue culture media forthese cell types.

[0132] As a ligand, the activity of z219a polypeptide can be measured bya silicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying adherent or non-adherenteukaryotic or prokaryotic cells. By measuring extracellularacidification changes in cell media over time, the microphysiometerdirectly measures cellular responses to various stimuli, including z219apolypeptide, its agonists, or antagonists. Preferably, themicrophysiometer is used to measure responses of a z219a-responsiveeukaryotic cell, compared to a control eukaryotic cell that does notrespond to z219a polypeptide. Z219A-responsive eukaryotic cells comprisecells into which a receptor for z219a has been transfected creating acell that is responsive to z219a; or cells naturally responsive to z219asuch as cells derived from, for example, pancreas, intestinal, prostateor tracheal tissue. Differences, measured by a change, for example, anincrease or diminution in extracellular acidification, in the responseof cells exposed to z219a polypeptide, relative to a control not exposedto z219a, are a direct measurement of z219a-modulated cellularresponses. Moreover, such z219a-modulated responses can be assayed undera variety of stimuli. Using the microphysiometer, there is provided amethod of identifying agonists of z219a polypeptide, comprisingproviding cells responsive to a z219a polypeptide, culturing a firstportion of the cells in the absence of a test compound, culturing asecond portion of the cells in the presence of a test compound, anddetecting a change, for example, an increase or diminution, in acellular response of the second portion of the cells as compared to thefirst portion of the cells. The change in cellular response is shown asa measurable change extracellular acidification rate. Moreover,culturing a third portion of the cells in the presence of z219apolypeptide and the absence of a test compound can be used as a positivecontrol for the z219a-responsive cells, and as a control to compare theagonist activity of a test compound with that of the z219a polypeptide.Moreover, using the microphysiometer, there is provided a method ofidentifying antagonists of z219a polypeptide, comprising providing cellsresponsive to a z219a polypeptide, culturing a first portion of thecells in the presence of z219a and the absence of a test compound,culturing a second portion of the cells in the presence of z219a and thepresence of a test compound, and detecting a change, for example, anincrease or a diminution in a cellular response of the second portion ofthe cells as compared to the first portion of the cells. The change incellular response is shown as a measurable change extracellularacidification rate. Antagonists and agonists, for z219a polypeptide, canbe rapidly identified using this method.

[0133] Moreover, z219a can be used to identify cells, tissues, or celllines which respond to a z219a-stimulated pathway. The microphysiometer,described above, can be used to rapidly identify ligand-responsivecells, such as cells responsive to z219a of the present invention. Cellscan be cultured in the presence or absence of z219a polypeptide. Thosecells which elicit a measurable change in extracellular acidification inthe presence of z219a are responsive to z219a. Such cell lines, can beused to identify antagonists and agonists of z219a polypeptide asdescribed above. z219a can also be used to identify inhibitors(antagonists) of its activity. Test compounds are added to the assaysdisclosed herein to identify compounds that inhibit the activity ofz219a. In addition to those assays disclosed herein, samples can betested for inhibition of z219a activity within a variety of assaysdesigned to measure receptor binding or the stimulation/inhibition ofz219a-dependent cellular responses. For example, z219a-responsive celllines can be transfected with a reporter gene construct that isresponsive to a z219a-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a z219a-DNA response element operably linked to a gene encodingan assayable protein, such as luciferase. DNA response elements caninclude, but are not limited to, cyclic AMP response elements (CRE),hormone response elements (HRE) insulin response element (IRE) (Nasrinet al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum responseelements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP responseelements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990.Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989.Candidate compounds, solutions, mixtures or extracts are tested for theability to inhibit the activity of z219a on the target cells asevidenced by a decrease in z219a stimulation of reporter geneexpression. Assays of this type will detect compounds that directlyblock z219a binding to cell-surface receptors, as well as compounds thatblock processes in the cellular pathway subsequent to receptor-ligandbinding. In the alternative, compounds or other samples can be testedfor direct blocking of z219a binding to receptor using z219a tagged witha detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC,and the like). Within assays of this type, the ability of a test sampleto inhibit the binding of labeled z219a to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

[0134] A z219a polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an F_(C) fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to (any specific uses?,affinity purify receptor, in vitro assay tool, antagonist). For use inassays, the chimeras are bound to a support via the F_(C) region andused in an ELISA format.

[0135] Z219a polypeptides can also be used to prepare antibodies thatspecifically bind to z219a epitopes, peptides or polypeptides. The z219apolypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. One of skill in theart would recognize that antigens or immunogenic epitopes can consist ofstretches of amino acids within a longer polypeptide from less thanabout 10 amino acids or longer, and up to about the entire length of thepolypeptide or longer depending on the polypeptide. Suitable antigensinclude the z219a polypeptide encoded by SEQ ID NO:2 from amino acidresidue 26 (Tyr) to residue 235 (Ser), or a contiguous 9 to 210 aminoacid fragment thereof. Moreover suitable antigens include Block 1, 2 or3, and motifs 1 through 5, disclosed herein. Preferred peptides to useas antigens are hydrophilic peptides such as those predicted by one ofskill in the art from a hydrophobicity plot, determined for example,from a Hopp/Woods hydrophilicity profile based on a sliding six-residuewindow, with buried G, S, and T residues and exposed H, Y, and Wresidues ignored. Antibodies generated from this immune response can beisolated and purified as described herein. Methods for preparing andisolating polyclonal and monoclonal antibodies are well known in theart. See, for example, Current Protocols in Immunology, Cooligan, et al.(eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995;Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

[0136] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a z219a polypeptide or a fragment thereof.The immunogenicity of a z219a polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of z219a polypeptideor a portion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like” , suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0137] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

[0138] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to z219a proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledz219a protein or peptide). Genes encoding polypeptides having potentialz219a polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.)Random peptide display libraries can be screened using the z219asequences disclosed herein to identify proteins which bind to z219a.These “binding proteins” which interact with z219a polypeptides can beused for tagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding proteins can also beused in analytical methods such as for screening expression librariesand neutralizing activity. The binding proteins can also be used fordiagnostic assays for determining circulating levels of polypeptides;for detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. These binding proteins can also act asz219a “antagonists” to block z219a binding and signal transduction invitro and in vivo. These anti-z219a binding proteins would be useful,for example, as antagonists for inhibiting z219a ligand from interactingwith its receptor.

[0139] Antibodies are determined to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with known related polypeptide molecules.First, antibodies herein specifically bind if they bind to a z219apolypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-z219a) polypeptide. Itis preferred that the antibodies exhibit a binding affinity (K_(a)) of10⁶ M⁻¹ or greater, preferably 10⁷ M⁻⁷ or greater, more preferably 10⁸M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater. The bindingaffinity of an antibody can be readily determined by one of ordinaryskill in the art, for example, by Scatchard analysis (Scatchard, G.,Ann. NY Acad. Sci. 51: 660-672, 1949).

[0140] Second, antibodies are determined to specifically bind if they donot significantly cross-react with known related polypeptides.Antibodies do not significantly cross-react with related polypeptidemolecules, for example, if they detect z219a but not known relatedpolypeptides using a standard Western blot analysis (Ausubel et al.,ibid.). Examples of known related polypeptides are orthologs, proteinsfrom the same species that are members of a protein family such as otherknown 2-19 family members, mutant z219a polypeptides, and non-humanz219a. Moreover, antibodies may be “screened against” known relatedpolypeptides to isolate a population that specifically binds to theinventive polypeptides. For example, antibodies raised to z219a areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to z219a will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art. See, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.

[0141] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to z219a proteinsor peptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant z219aprotein or polypeptide.

[0142] Antibodies to z219a may be used for tagging cells that expressz219a; for isolating z219a by affinity purification; for diagnosticassays for determining circulating levels of z219a polypeptides; fordetecting or quantitating soluble z219a as marker of underlyingpathology or disease; in analytical methods employing FACS; inanalytical methods employing in situ hybridization and histology; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockz219a activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to z219apolypeptide or fragments thereof may be used in vitro to detectdenatured z219a polypeptide or fragments thereof in assays, for example,Western Blots or other assays known in the art.

[0143] Antibodies or polypeptides herein can also be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, z219apolypeptides or anti-z219a antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe anti-complementary molecule.

[0144] Suitable detectable molecules may be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Polypeptides or antibodies may also be conjugated tocytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the polypeptide or antibodyportion. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0145] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0146] In another embodiment, z219a-cytokine fusion proteins orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood and bone marrow cancers),if the z219a polypeptide or anti-z219a antibody targets thehyperproliferative blood or bone marrow cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable z219a polypeptidesor anti-z219a antibodies target an undesirable cell or tissue (i.e., atumor or a leukemia), and the fused cytokine mediated improved targetcell lysis by effector cells. Suitable cytokines for this purposeinclude interleukin 2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), for instance.

[0147] In yet another embodiment, the z219a polypeptide or anti-z219aantibody can target vascular cells or tissues. Such polypeptide orantibody can be conjugated with a radionuclide, and particularly with abeta-emitting radionuclide, to reduce restenosis. Such therapeuticapproach poses less danger to clinicians who administer the radioactivetherapy. For instance, iridium-192 impregnated ribbons placed intostented vessels of patients until the required radiation dose wasdelivered showed decreased tissue growth in the vessel and greaterluminal diameter than the control group, which received placebo ribbons.Further, revascularisation and stent thrombosis were significantly lowerin the treatment group. Similar results are predicted with targeting ofa bioactive conjugate containing a radionuclide, as described herein.

[0148] The bioactive polypeptide or antibody conjugates described hereincan be delivered intravenously, intraarterially or intraductally, or maybe introduced locally at the intended site of action.

[0149] Molecules of the present invention can be used to identify andisolate receptors for z219a. For example, proteins and peptides of thepresent invention can be immobilized on a column and membranepreparations run over the column (Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp.195-202). Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification” , M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-1180,1984) and specific cell-surfaceproteins can be identified.

[0150] The polypeptides, nucleic acid and/or antibodies of the presentinvention can be used in treatment of disorders associated with type Iand type II diabetes, gestational diabetes, pancreatic cancer, nutrientand metabolic disorders, pancreatic and intestinal hormonal release,intestinal mucosal secretion, intestinal regeneration from acute injury,peptic ulcers, Crohn's disease, inflammatory bowel disease, defense ofthe GI tract against microbial attack, other epithelial disorders, andprostate obstruction and cancer. The molecules of the present inventioncan be used to modulate other proteins to which they interact, or totreat or prevent development of pathological conditions in such diversetissues as small intestine, pancreas, and prostate. In particular,certain diseases such as diabetes, peptic ulcers, Crohn's disease,inflammatory bowel disease, certain genetic syndromes and other humandiseases may be amenable to such diagnosis, treatment or prevention.

[0151] Polynucleotides encoding z219a polypeptides are useful withingene therapy applications where it is desired to increase or inhibitz219a activity. If a mammal has a mutated or absent z219a gene, thez219a gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a z219a polypeptide is introduced in vivo ina viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0152] In another embodiment, a z219a gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0153] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0154] Antisense methodology can be used to inhibit z219a genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a z219a-encodingpolynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:l) aredesigned to bind to z219a-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof z219a polypeptide-encoding genes in cell culture or in a subject.

[0155] The present invention also provides reagents which will find usein diagnostic applications. For example, the z219a gene, a probecomprising z219a DNA or RNA or a subsequence thereof can be used todetermine if the z219a gene is present on chromosome 21 or if a mutationhas occurred. The polynucleotides of the present invention map to the21q22.3 region on human chromosome 21 (see, Example 3). Polynucleotidesof the present invention are also used to detect abnormalities on humanchromosome 21 associated with disease or other human traits. Detectablechromosomal aberrations at the z219a gene locus include but are notlimited to aneuploidy, gene copy number changes, insertions, deletions,restriction site changes and rearrangements. Such aberrations can bedetected using polynucleotides of the present invention by employingmolecular genetic techniques, such as restriction fragment lengthpolymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel, et. al., ibid.;Marian, A. J., Chest, 108: 255-265, 1995). These methods can be employedto use z219a polynucleotides to detect abnormalities on human chromosome21, such as those described below.

[0156] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0157] The z219a gene is located at the 21q22.3 region of chromosome 21.Trisomy 21, one of the most common chromosomal abnormalities, causesDown Syndrome (Penrose, L. S., J. Genet. 27:219, 1933; Hook, E. G., J.Am. Med. Assoc. 249:2034-2038, 1983). Moreover, the Down Syndromecritical region is the 21q22.3 locus of chromosome 21. Thus, since thez219a gene maps to this critical region, the z219a polynucleotide probesof the present invention can be used to detect Down Syndrome trisomy orpartial trisomy (Rahmani, Z. et al., Proc. Nat. Acad. Sci. 86:5958-5962,1989; Delabar, J. M. et al., Europ. J. Hum. Genet. 1:114-124, 1993).Moreover, several genes of known function map to this region. Forexample, the trefoil factors map to the 21q22.3 region, as discussedherein. Moreover, amongst other genetic loci, those for KnoblochSyndrome (21q22.3); collagen IV (alpha-1 and -2) and collagen VIIIalpha-1 (21q22.3); integrin beta-2 (21q22.3); interferon receptors(21q22.1); and familial platelet disorder (21q22.1-q22.2), all manifestthemselves in human disease states as well as map to this region of thehuman genome. See the Online Mendellian Inheritance of Man (OMIM) genemap, and references therein, for this region of chromosome 21 on apublicly available WWW server(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=21q22.3).All of these serve as possible candidate genes for an inheritabledisease which show linkage to the same chromosomal region as the z219agene.

[0158] Similarly, defects in the z219a gene itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a z219a geneticdefect.

[0159] Mice engineered to express the z219a gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of z219agene function, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express z219a, either ubiquitously or under atissue-specific or tissue-restricted promoter can be used to ask whetherover-expression causes a phenotype. For example, over-expression of awild-type z219a polypeptide, polypeptide fragment or a mutant thereofmay alter normal cellular processes, resulting in a phenotype thatidentifies a tissue in which z219a expression is functionally relevantand may indicate a therapeutic target for the z219a, its agonists orantagonists. For example, a preferred transgenic mouse to engineer isone that over-expresses the mature z219a polypeptide. Moreover, suchover-expression may result in a phenotype that shows similarity withhuman diseases. Similarly, knockout z219a mice can be used to determinewhere z219a is absolutely required in vivo. The phenotype of knockoutmice is predictive of the in vivo effects of that a z219a antagonist,such as those described herein, may have. The human z219a cDNA can beused to isolate murine z219a mRNA, cDNA and genomic DNA, which aresubsequently used to generate knockout mice. These mice may be employedto study the z219a gene and the protein encoded thereby in an in vivosystem, and can be used as in vivo models for corresponding humandiseases. Moreover, transgenic mice expression of z219a antisensepolynucleotides or ribozymes directed against z219a, described herein,can be used analogously to knockout mice described above.

[0160] Other diagnostic applications using z219a can be employed. Forexample, the z219a gene, a probe comprising z219a DNA or RNA or asubsequence thereof can be used to determine if the z219a gene isexpressed differently in diseased tissues. For example, among otherdiseases, z219a may be expressed in certain pancreatic, prostatic,intestinal, throat and lung cancers, or other diseases associated withthose tissues. In the alternative, z219a expression in certain tissuesmay be decreased in certain disease states relative to normal.

[0161] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified z219a polypeptide incombination with a pharmaceutically acceptable vehicle. Thispharmaceutical composition will be used to modulate energy balance inmammals or to protect epithelial cells from injury.

[0162] With regard to modulating energy balance, z219a polypeptidesmodulate cellular metabolic reactions. Such metabolic reactions includeadipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucoseuptake, protein synthesis, thermogenesis, oxygen utilization and thelike. The expression pattern of z219a polypeptide indicates expressionin epithelial cell tissues. With regard to epithelial cell protection,z219a polypeptide may be used in organ preservation, forcryopreservation, for surgical pretreatment to prevent injury due toischemia and/or inflammation or in like procedures. In this regard,z219a polypeptides may find utility in modulating nutrient uptake, asdemonstrated, for example, by 2-deoxy-glucose uptake in the brain or thelike.

[0163] The z219a polypeptides may modulate mammalian energy balance. Theexpression pattern of z219a polypeptide indicates expression inpancreas. Among other methods known in the art or described herein,mammalian energy balance may be evaluated by monitoring one or more ofthe following metabolic functions: adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, protein synthesis,thermogenesis, oxygen utilization or the like. These metabolic functionsare monitored by techniques (assays or animal models) known to one ofordinary skill in the art, as is more fully set forth below. Forexample, the glucoregulatory effects of insulin are predominantlyexerted in the liver, skeletal muscle and adipose tissue. Insulin bindsto its cellular receptor in these three tissues and initiatestissue-specific actions that result in, for example, the inhibition ofglucose production and the stimulation of glucose utilization. In theliver, insulin stimulates glucose uptake and inhibits gluconeogenesisand glycogenolysis. In skeletal muscle and adipose tissue, insulin actsto stimulate the uptake, storage and utilization of glucose.

[0164] Moreover, pancreatic expression of z219a polypeptide suggeststhat pharmaceutical compositions of the present invention may be usefulin prevention or treatment of pancreatic disorders associated withpathological regulation of the expansion of neurocrine and exocrinecells in the pancreas, such as IDDM, pancreatic cancer or the like.Pharmaceutical compositions of the present invention may also beinvolved in prevention or treatment of pancreatic conditionscharacterized by dysfunction associated with pathological regulation ofblood glucose levels, insulin resistance or digestive function.

[0165] Art-recognized methods exist for monitoring all of the metabolicfunctions recited above. Thus, one of ordinary skill in the art is ableto evaluate z219a polypeptides, fragments, fusion proteins, antibodies,agonists and antagonists for metabolic modulating functions. Exemplarymodulating techniques are set forth below.

[0166] Adipogenesis, gluconeogenesis and glycogenolysis are interrelatedcomponents of mammalian energy balance, which may be evaluated by knowntechniques using, for example, ob/ob mice or db/db mice. The ob/ob miceare inbred mice that are homozygous for an inactivating mutation at theob (obese) locus. Such ob/ob mice are hyperphagic and hypometabolic, andare believed to be deficient in production of circulating OB protein.The db/db mice are inbred mice that are homozygous for an inactivatingmutation at the db (diabetes) locus. The db/db mice display a phenotypesimilar to that of ob/ob mice, except db/db mice display a more severediabetic phenotype. Such db/db mice are believed to be resistant to theeffects of circulating OB protein. Also, various in vitro methods ofassessing these parameters are known in the art.

[0167] Insulin-stimulated lipogenesis, for example, may be monitored bymeasuring the incorporation of ¹⁴C-acetate into triglyceride (Mackall etal. J. Biol. Chem. 251:6462-6464, 1976) or triglyceride accumulation(Kletzien et al., Mol. Pharmacol. 41:393-398, 1992).

[0168] Glucose uptake may be evaluated, for example, in an assay forinsulin-stimulated glucose transport. Non-transfected, differentiated L6myotubes (maintained in the absence of G418) are placed in DMEMcontaining 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mMglutamine. After two to five hours of culture, the medium is replacedwith fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulinor IGF-1, or a dilution series of the test substance, are added, and thecells are incubated for 20-30 minutes. ³H or ¹⁴C-labeled deoxyglucose isadded to ≈50 1 M final concentration, and the cells are incubated forapproximately 10-30 minutes. The cells are then quickly rinsed with coldbuffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDSor 1 N NaOH). The cell lysate is then evaluated by counting in ascintillation counter. Cell-associated radioactivity is taken as ameasure of glucose transport after subtracting non-specific binding asdetermined by incubating cells in the presence of cytochalasin b, aninhibitor of glucose transport. Other methods include those describedby, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol.Metab. 29) :E326-E333, 1994 (insulin-stimulated glucose transport).

[0169] Protein synthesis may be evaluated, for example, by comparingprecipitation of ³⁵S-methionine-labeled proteins following incubation ofthe test cells with ³⁵S-methionine and ³⁵S-methionine and a putativemodulator of protein synthesis.

[0170] Thermogenesis may be evaluated as described by B. Stanley in TheBiology of Neuropeptide Y and Related Peptides, W. Colmers and C.Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C.Billington et al., Am. J. Physiol. 260:R321, 1991; N. Zarjevski et al.,Endocrinology 133:1753, 1993; C. Billington et al., Am. J. Physiol.266:R1765, 1994; Heller et al., Am. J. Physiol. 252(4 Pt 2): R661-7,1987; and Heller et al., Am. J. Physiol. 245(3): R321-8, 1983. Also,metabolic rate, which may be measured by a variety of techniques, is anindirect measurement of thermogenesis.

[0171] Oxygen utilization may be evaluated as described by Heller etal., Pflugers Arch 369(1): 55-9, 1977. This method also involved ananalysis of hypothalmic temperature and metabolic heat production.Oxygen utilization and thermoregulation have also been evaluated inhumans as described by Haskell et al., J. Appl. Physiol. 51(4): 948-54,1981.

[0172] The z219a polypeptide is expressed in the small intestine,pancreas and salivary glands. Secreted products from those organs canplay important roles in the maintenance of normal gastric epithelium andfunction. Thus, z219a polypeptide pharmaceutical compositions of thepresent invention may also be useful in prevention or treatment ofgastric mucositis. Mucositis is manifested by the damage and loss ofintegrity of the oral and gastric epithelium. Such damage often providesa microbial port of entry leading to sepsis. Mucositis is often inducedby chemotherapy and radiation therapy, and is often a dose-limiting sideeffect as well as a cause of mortality in cancer patients undergoingsuch treatment. The z219a polypeptides of the present invention mayprovide protection against gastric mucositis, analogous to some growthfactors and cytokines, for example, interleukin-11 (Orazi, A. et al.,Lab. Invest. 75:33-42, 1996). The effect of z219a prevention ortreatment of gastric mucositis can be measured in in vivo animal models,for example, the Syrian hamster model or in murine models using methodsdescribed in the art (Sonis, S. T. et al., Oral Surg. Oral Med. OralPathol. 69:437-443, 1990; Farrell, C. L. et al., Cancer Res. 58:933-939,1998; Orazi, A. et al., supra.).

[0173] The z219a polypeptide is expressed in the small intestine. Thus,z219a polypeptide pharmaceutical compositions of the present inventionmay also be useful in prevention or treatment of digestive disorders inthe GI tract, such as disorders associated with pathological secretorycell expansion or differentiation. Assays and animal models are known inthe art for monitoring such expansion or differentiation and forevaluating z219a polypeptide, fragment, fusion protein, antibody,agonist or antagonist in the prevention or treatment thereof.

[0174] Moreover, trefoil factors in the intestine are known to beinvolved in mucosal stabilization in the gut and repair processesassociated with acute injury particularly epithelial restitution(Poulsom, R., Bail. Clin. Gastro., 10; 113-134, 1996; Sands, B. E., andPodolsky, D. K., Annu. Rev. Phvsiol., 58; 253-273, 1996. Also, trefoilproteins appear to have a role in healing wounds caused by intestinalinflammatory diseases, and resisting microbial invasion via mucosalsecretion involvement (Palut, A. G., New Eng. J. Med., 336; 5-6-507,1997; Playford, R. J., J. Royal Coll. Phys. London, 31; 37-41, 1997)Epidermal growth factor (EGF) receptor ligands may play a role inenhancing trefoil activity in the gut; however, repair of mucosal injuryis not dependent in the main endogenous EGF receptor ligand in the gut,TNF-α, suggesting a role of other undiscovered ligands (Cook, G. A., etal., Am. Physiol. Soc., G1540-G1549, 1997). For example, the z219apolypeptides may serve as such ligand, regulatory protein or otherfactor in the trefoil pathway, and hence play an important therapeuticrole in diseases and injury associated with the gut and mucosalepithelium.

[0175] Moreover, mapping data disclosed herein (Example3) shows thatz219a maps on chromosome 21 near trefoil factor 3 and spasmolyticprotein 1, both GI peptides. Thus, z219a is also useful as a marker forthese factors and diseases associated with them.

[0176] Also, z219a polypeptide is expressed in the pancreas polypeptideand may be independent of gastrointestinal function. Thus, z219apolypeptide pharmaceutical compositions of the present invention may beuseful in prevention or treatment of pancreatic disorders associatedwith pathological regulation of the expansion of neuroendocrine andexocrine cells in the pancreas, such as IDDM, pancreatic cancer,pathological regulation of blood glucose levels, insulin resistance ordigestive function.

[0177] The z219a polypeptide of the present invention may act in theneuroendocrine/exocrine cell fate decision pathway and is thereforecapable of regulating the expansion of neuroendocrine and exocrine cellsin the pancreas. One such regulatory use is that of islet cellregeneration. Also, it has been hypothesized that the autoimmunity thattriggers IDDM starts in utero, and z219a polypeptide is a developmentalgene involved in cell partitioning. Assays and animal models are knownin the art for monitoring the exocrine/neuroendocrine cell lineagedecision, for observing pancreatic cell balance and for evaluating z219apolypeptide, fragment, fusion protein, antibody, agonist or antagonistin the prevention or treatment of the conditions set forth above.

[0178] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified z219a polypeptide incombination with a pharmaceutically acceptable vehicle. Suchpharmaceutical compositions may be administered to prevent or treatsalivary gland dysfunction. Such prevention or treatment may be directedto digestive dysfunction, such as a deficiency in starch breakdowncapability or efficiency, wound healing dysfunction, inadequate salivaproduction or composition or mucosal integrity breakdown. Z219apolypeptides may also have an anti-microbial function. Also, expressionof z219a polypeptide at a relatively high level in trachea may indicatea role for z219a polypeptides in prevention or treatment of destructivelung disease. Examples of pathological conditions, characterized by oneor more of the aforementioned criteria, include xerostomia, sarcoidosis,dental caries, osteomyelitis, oral candidiasis, buccal mucosainfections, chronic inflammation (Sjogren's syndrome), mumps, chronicbronchitis, adult respiratory distress syndrome (ARDS), sudden infantdeath syndrome (SIDS), salivary gland carcinoma, pneumocystic carinii(particularly as associated with AIDS patients), cystic fibrosis,emphysema and the like.

[0179] Evaluation of z219a polypeptide involvement in such conditionsmay be conducted using in vivo or in vitro methods that are known tothose of ordinary skill in the art. For example, bronchoalveolar lavagemay be employed in the assessment of destructive lung diseases, such aspulmonary emphysema, chronic bronchitis, cystic fibrosis, ARDS and thelike. See, for example, Luisetti et al., Respiration 59(suppl. 1):24-27, 1992. Salivary gland, lacrimal gland and labial salivary glandbiopsies may be employed in the evaluation of xerostomia. See, forexample, Matsumoto et al., J. Clin. Invest. 97(8): 1969-77, 1996. Thiscalcium channel dependent condition has also been evaluated using fura-2assays of intracellular calcium ion concentration, as described inSeagrave et al., Archs. Oral Biol. 41(5): 425-30, 1996. Alymphoplasia(aly) mice are a useful animal model for systemic Sjogren's syndrome, anautoimmune disease characterized by lymphocytic infiltration into thelachrymal and salivary glands, leading to symptomatic dry eyes andmouth. See, for example, Furukawa et al., British Journal ofRheumatology 35: 1223-30, 1996.

[0180] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. For applications for which local effects are preferred,such as for influencing the formation of certain types of mature cellsfrom localized (e.g., pancreatic) stem cells, formulations designed forlocal administration are preferred. Such pharmaceutical compositions areamenable, for example, to implantation or other local delivery methodand may additionally be formulated for sustained release. Formulation ofpharmaceutical compositions for a variety of modes of administration iswithin the ordinary skill in the art. In general, pharmaceuticalformulations will include a z219a protein in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally bedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. The proteins may be administered for acute treatment, overone week or less, often over a period of one to three days or may beused in chronic treatment, over several months or years.

[0181] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Extension of EST Sequence

[0182] Scanning of a translated DNA database using a human proteinscontaining a signal sequence as a query resulted in identification of anexpressed sequence tag (EST) sequence found to be homologous to thehuman 2-19 protein sequence and designated z219a.

[0183] Confirmation of the EST sequence was made by sequence analyses ofthe cDNA from which the EST originated. This cDNA was contained in aplasmid, and was excised using Eco RI and Not I cloning sites. The cDNAclone was sequenced using the following primers: ZC 695 (SEQ ID NO:9),ZC 7231 (SEQ ID NO:10), and ZC 13,695 (SEQ ID NO:11), ZC 13,789 (SEQ IDNO:12), and ZC 13,790 (SEQ ID NO:13). Sequencing results indicated a 946bp insert with a 707 bp open reading frame beginning with an initiatingMet and ending with a stop signal. The sequence analyses revealed thatthe cDNA encompassed the entire coding region of the DNA encoding z219a.

Example 2 Tissue Distribution

[0184] Northerns were performed using Human Multiple Tissue Blots fromClontech (Palo Alto, Calif.). Using oligonucleotide ZC 14,069 (SEQ IDNO:14) and oligonucleotide ZC 13,072 (SEQ ID NO:15) as primers, aninternal segment of z219a was reamplified. The PCR reaction conditionswere as follows: an initial 1 cycle 5 minute denaturation at 94° C.; 30cycles of a 30 second denaturation at 94° C., 30 second annealing andextension at 60° C.; followed by a 4° C. soak. The resulting PCR productwas electrophoresed on a 1% agarose gel, the 300 bp fragment waspurified using a QIAquick column (Qiagen, Inc., Chatsworth, Calif.) andthen radioactively labeled using a random priming REDIPRIME DNA labelingsystem (Amersham, Arlington Heights, Ill.) according to themanufacturer's specifications. The probe was purified using a NUCTRAPpush column (Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB(Clontech) solution was used for prehybridization and as a hybridizationsolution for the Northern blots. Hybridization took place overnight at50° C., and the blots were then washed in 2×SSC and 0.05% SDS at roomtemperature, followed by a wash in 1×SSC and 0.1% SDS at 50° C. Onetranscript size was observed at approximately 1 kb. Signal intensity washighest for pancreas, with moderately strong signals in small intestine,and prostate. Relatively less intense signals were present in placenta,kidney, testis, stomach, thyroid and trachea A weaker signal was presentin lung.

[0185] Dot Blots were also performed using Human RNA Master Blots™(Clontech). The methods and conditions for the Dot Blots are the same asfor the Multiple Tissue Blots disclosed above. Strong signal intensitywas present in salivary gland, pancreas, and bone marrow. Less intensesignals were indicated in prostate, stomach, small intestine, thymus,trachea, placenta, fetal kidney, and fetal liver.

[0186] Northern analysis was performed using a Human Fetal MTN II™ Blot(Clontech). A probe was obtained by PCR using primers ZC14,069 (SEQ IDNO:14) and ZC14,072 (SEQ ID NO:15) with the cDNA described in Example 1as a template. PCR reaction conditions were as follows: one cycle at 94°C. for 1.5 minutes; 25 cycles at 94° C. for 10 seconds, 60° C. for 20seconds, 72° C. for 30 seconds; one cycle at 72° C. for 5 minutes;followed by 4° C. hold. A sample of the reaction mixture waselectrophoresed to confirm amplification of a 330 bp fragment, and theremainder was purified on a Microspin-300 column (Pharmacia). The probewas radioactively labeled with ³²P-dCTP using Rediprime Labeling System(Amersham) according to manufacturer's instructions. The probe waspurified using a NUCTRAP push column(Stratagene). EXPRESSHYB solution(Clontech) was used for prehybridization and hybridization. Thehybridization solution consisted of 8 ml EXPRESSHYB, 80 μl shearedsalmon sperm DNA (10 mg/ml; 5 Prime-3 Prime, Boulder, Colo.), 48 μlHuman Cot-1 DNA (1 mg/ml; GibcoBRL) and 12 μl labeled probe (1.5×10⁶CPM/μl) . Hybridization took place overnight at 55° C. The blots werewashed in 2×SSC/0.1% SDS at room temperature, then 2×SSC/0.1% SDS at 65°C., followed by 1×SSC/0.1% SDS wash at 65° C. A faint signal atapproximately 1 kb was observed in fetal kidney.

Example 3 PCR-Based Chromosomal Mapping of the z219a Gene

[0187] Z219a was mapped to chromosome 21 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontains DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available WWWserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

[0188] For the mapping of z219a with the “GeneBridge 4 RH Panel” , 20 ρlPCR reactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2 μl 1×KlenTaqPCR reaction buffer (Clontech), 1.6 μl dNTPs mix (2.5 mM each,Perkin-Elmer, Foster City, Calif.), 1 μl sense primer, ZC13,956 (SEQ IDNO:16), 1 μl antisense primer, ZC13,957 (SEQ ID NO:17), 2 μl “RediLoad”(Research Genetics, Inc.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix(Clontech), 25 ng of DNA from an individual hybrid clone or control andddH₂O for a total volume of 20 μl. The reactions were overlaid with anequal amount of mineral oil and sealed. The PCR cycler conditions wereas follows: an initial 1 cycle 5 minute denaturation at 95° C.; 35cycles of a 1 minute denaturation at 95° C., 1 minute annealing at 62°C. and 1.5 minute extension at 72° C.; followed by a final 1 cycleextension of 7 minutes at 72° C. The reactions were separated byelectrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts,Rockland, Me.).

[0189] The results showed that z219a maps 210.83 cR_(—)3000 from the topof the human chromosome 21 linkage group on the WICGR radiation hybridmap. Proximal and distal framework markers were D21S1826 and D21S266,respectively. The use of surrounding markers positions z219a in the21q22.3 region on the integrated LDB chromosome 21 map (The GeneticLocation Database, University of Southhampton, WWW server:http://cedar.genetics. soton.ac.uk/public_html/)

Example 4 Construction of z219a Mammalian Expression Vectorz219aCEE/pZP9

[0190] An expression vector was prepared for the z219a polypeptide,z2l9aCEE/pZP9, wherein the construct is designed to express a z219apolypeptide with a C-terminal Glu-Glu tag (SEQ ID NO:18).

[0191] A 730 bp PCR generated z219a DNA fragment was created usingZC14,870 (SEQ ID NO:19) and ZC15,101 (SEQ ID NO:20) as PCR primers toadd EcoRI and BamHI restriction sites. The cDNA, described above inexample 1, was used as a template. PCR amplification of the z2l9afragment was performed as follows: one cycle at 94° C. for 1.5 minutes;five cycles at 94° C. for 10 seconds, 30° C. for 20 seconds, 72° C. for1 minute; twenty-five cycles at 94° C. for 10 seconds, 65° C. for 20seconds, 72° C. for 1 minute; one cycle at 72° C. for 5 minutes;followed by a 4° C. hold. The reaction was purified on a Microspin-400cloumn (Pharmacia) and digested with EcoRI and BamHI (Gibco ERL)following manufacturer's protocol. A band of the predicted size, 730 bp,was visualized by 1% agarose gel electrophoresis, excised and the DNAwas purified using a QiaexII™ purification system (Qiagen) according themanufacturer's instructions.

[0192] The excised DNA was subcloned into plasmid CEEpZP9 which had beencut with BamHI and EcoRI. The z219aCEE/pZP9 expression vector uses thenative z219a signal peptide and attaches the Glu-Glu tag (SEQ ID NO:21)to the C-terminus of the z219a polypeptide-encoding polynucleotidesequence. Plasmid pZP9 (deposited at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., ATCC No. 98668) is amammalian expression vector containing an expression cassette having themouse metallothionein-1 promoter, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.

[0193] About 30 ng of the restriction digested C-terminal Glu-Glu-z219ainsert and about 12 ng of the digested vector were ligated overnight at16° C. One microliter of each ligation reaction was independentlyelectroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 50 μg/ml ampicillin, and incubated overnight. Colonies werescreened by PCR using primers ZC13,006 (SEQ ID NO:22) and ZC13,007 (SEQID NO:23). PCR screening was done at 94° C. for 4 minutes; 25 cycles of94° C. for 10 seconds, 58° C. for 20 seconds, 72° C. for 1 minute;followed by 72° C. for 5 minutes. Positive clones were plated on to LBAmp plates. The insert sequence of positive clones was verified bysequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

Example 5 Transfection and Expression of z219aCEE Polypeptides

[0194] BHK 570 cells (ATCC No. CRL-10314) were plated in 10 cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 2 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid z219aCEE/pZP9 (see, Example 4), using Lipofectamine™ (GibcoBRL), in serum free (SF) media formulation (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 2 mM L-glutamine, 2 mM sodiumpyruvate, 10 ug/ml transferrin, 5 mg/ml insulin, 10 mg/ml fetuin and 2ng/ml selenium). Sixteen micrograms of z219aCEE/pZP9 were separatelydiluted into 15 ml tubes to a total final volume of 640 ml SF media. Inseparate tubes, 35 ml of Lipofectamine™ (Gibco BRL) was mixed with 605ml of SF medium. The Lipofectamine™ mix was added to the DNA mix andallowed to incubate approximately 30 minutes at room temperature. Fivemilliliters of SF media was added to the DNA:Lipofectamine™ mixture. Thecells were rinsed once with 5 ml of SF media, aspirated, and theDNA:Lipofectamine™ mixture was added. The cells were incubated at 37° C.for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added tothe plate. The plate was incubated at 37° C. overnight and theDNA:Lipofectamine™ mixture was replaced with fresh FBS/DMEM media thenext day. On day 2 post-transfection, the cells were split into theselection media (ESTEP #1 with 1 mM MTX) in 150 mm plates at 1:50, 1:100and 1:200. The plates were refed at day 5 post-transfection with freshselection media.

[0195] Approximately 10-12 days post-transfection, one 150 mm culturedish of methotrexate resistant colonies was chosen from eachtransfection, the media aspirated, the plates washed with 10 mlserum-free ESTEP 2 media (668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvicacid, sodium salt 96% (Mallinckrodt), 185.0 g/50L NaHCO₃ (Mallinkrodt),5.0 mg/ml, 25 ml/50L insulin, 10.0 mg/ml and 25 ml/50 L transferrin).The wash media was aspirated and replaced with 5 ml serum-free ESTEP 2.Sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, Calif.)pre-soaked in serum-free ESTEP 2 was then placed over the cells. Asterile nitrocellulose filter pre-soaked in serum-free ESTEP 2 was thenplaced over the mesh. Orientation marks on the nitrocellulose weretransferred to the culture dish. The plates were then incubated for 5-6hours in a 37° C., 5% CO₂ incubator. Following incubation, the filterwas removed, and the media aspirated and replaced with DMEM/5% FBS,1×PSN (Gibco BRL) media. The filter was then placed into a sealable bagcontaining 50 ml buffer (25 mM Tris, 25 mM glycine, 5 mMβ-mercaptoethanol) and incubated in a 65° C. water bath for 10 minutes.The filters were blocked in 10% nonfat dry milk/Western A buffer(Western A: 50mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and0.25% gelatin) for 15 minutes at room temperature on a rotating shaker.The filter was then incubated with an anti-Glu-Glu antibody-HRPconjugate at a 1:1000 dilution in 2.5% nonfat dry milk/Western A buffer(Western A: 50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and0.25% gelatin) overnight at 4° C. on a rotating shaker. The filter wasthen washed three times at room temperature in PBS plus 0.1% Tween 20,5-15 minutes per wash. The filter was developed with ECL reagent(Amersham Corp., Arlington Heights, Ill.) according the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham) forapproximately 5 minutes.

[0196] The 150 mm culture dish was also trypsinized and the remainder ofthe cells were pooled and subjected to Western Blot analysis andmycoplasma testing. The pool was frozen for storage.

Example 6 Expression of z219aCEE in Baculovirus

[0197] A baculovirus expression vector, designated p219aCEE, wasprepared to express a Z219a polypeptide with a C-terminal Glu-Glu tag(SEQ ID NO:18) .in insect cells. A z219a fragment was generated byEcoRI/XbaI restriction digest of z219aCEE/pZP9 (Example 4). Theresulting 946 bp fragment was visualized by gel electrophoresis (1%SeaPlaque/1% NuSieve). The band was excised, diluted to 0.5% agarosewith 2 mM MgCl2, melted at 65° C. and ligated into a Eco RI/XbaIdigested baculovirus expression vector, pZBV4L (a modified pFastBac™expression vector (Life Technologies) containing the late activatingBasic Protein promoter. About 90 nanograms of the restriction digestedz2l9aCEE insert and about 150 ng of the digested vector were ligatedovernight. The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH7.5 and 1 mM EDTA) and 4 fmol of the diluted ligation mix wastransformed into DH5α Library Efficiency competent cells (LifeTechnologies) according to manufacturer's direction by heat shock for 45seconds in a 42° C. waterbath. The ligated DNA was diluted in 450 μl ofSOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl,1.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) and plated ontoLB plates containing 100 μg/ml ampicillin. Clones were analyzed byrestriction digests and 1 μl of the positive clone was transformed into20 μl DH10Bac Max Efficiency™ competent cells (GIBCO-BRL, Gaithersburg,Md.) according to manufacturer's instruction, by heat shock for 45seconds in a 42° C. waterbath. The ligated DNA was diluted in 980 μl SOCmedia (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) and plated ontoLuria Agar plates containing 50 μg/ml kanamycin, 7 μg/ml gentamycin, 10μg/ml tetracycline, IPTG and Bluo Gal. The cells were incubated for 48hours at 37° C. A color selection was used to identify those cellshaving virus that had incorporated into the plasmid (referred to as a“bacmid” ). Those colonies, which were white in color, were picked foranalysis. Bacmid DNA was isolated from positive colonies using theQiaVac™ Miniprep8 system (Qiagen) according the manufacturer'sdirections. Clones were screened for the correct insert by amplifyingDNA using primers to the Basic Protein promoter and to the SV40 terminusvia PCR. Those having the correct insert were used to transfectSpodoptera frugiperda (Sf9) cells.

[0198] To transfect p219aCEE into insect cells, Sf9 cells were seeded at5×10⁶ cells per 35 mm plate and allowed to attach for 1 hour at 27° C.Five microliters of p219aCEE bacmid DNA was diluted with 100 μl Sf-900II SFM. Six μl of CellFECTIN™ Reagent (Life Technologies) was dilutedwith 100 μl Sf-900 II SFM. The bacmid DNA and lipid solutions weregently mixed and incubated 30-45 minutes at room temperature. The mediafrom one plate of cells were aspirated, the cells were washed 1×with 2ml fresh media. Eight hundred microliters of Sf-900 II SFM was added tothe lipid-DNA mixture. The wash media was aspirated and the DNA-lipidmix added to the cells. The cells were incubated at 27° C. for 4-5hours. The DNA-lipid mix was aspirated and 2 ml of Sf-900 II media wasadded to each plate. The plates were incubated at 27° C., 90% humidity,for 96 hours after which the virus, designated Ac219aCEE, was harvested.

[0199] To amplify virus, Sf9 cells were grown in 50 ml Sf-900 II SFM ina 125 ml shake flask to an approximate density of 0.41-0.52×10⁵cells/ml. They were then infected with 100 μl of the virus stock fromabove and incubated at 27° C. for 3-4 days after which time the viruswas harvested. The titer for Ac219aCEE was 2.3×10⁷ pfu/ml. To scale up,five liters of SF 900 II SFM containing SF9 cells was incubated at 27°C. and grown for approximately 72 hours. The cells were then infectedwith the harvested virus, described above, (MOI 1-3) and incubated at27° C. for approximately 48 hours. The cell density at infection wasapproximately 1×10⁶ cells/ml.

Example 7 Purification of z2l9aCEE Polypeptide from Baculovirus InfectedSf9 Cells

[0200] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying z219a polypeptidecontaining C-terminal GluGlu (EE) tags. A Protease inhibitor solutionwas added to 2000 ml of conditioned media from Ac219aCEE-infected Sf9cells (see, Example 6) to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). The sample was centrifuged at 10,000 rpm for 30min. at 4° C. in a Beckman JLA-10.5 rotor (Beckman Instruments, PaloAlto, Calif.) in a Beckman Avanti J25I centrifuge (Beckman Instruments)to remove cell debris. To the supernatant fraction, 50.0 ml of anti-EESepharose (prepared as described below) was added, and the mixture wasgently agitated on a Wheaton (Millville, N.J.) roller culture apparatusfor 18.0 h at 4° C.

[0201] The mixture was poured into a 5.0×20.0 cm Econo-Column (Bio-Rad,Laboratories, Hercules, Calif.) and the anti-EE Sepharose gel was washedwith 30 column volumes of phosphate buffered saline (PBS). Theabsorbance at 280 nM of the unretained flow-through fraction wasmeasured until the absorbance was less than 0.05. The flow-throughfraction was discarded. Once the absorbance of the flow-through was lessthan 0.05, column flow rate was reduced to zero. The anti-EE Sepharosegel was then washed with 2.0 column volumes of PBS containing 0.2 mg/mlEE peptide (Anaspec, San Jose, Calif.) for 1.0 h at 4° C. The EE peptideused has the sequence N-GluTyrMetProValAsp-C (SEQ ID NO:24). After,washing the column flow was resumed and the eluted polypeptide,containing both z219aCEE polypeptide and EE peptide, was collected. Thisfraction is referred to as the “polypeptide elution fraction.” Theanti-EE Sepharose gel was washed with 2.0 column volumes of 0.1 Mglycine, pH 2.5, and the glycine wash was collected separately. The pHof the glycine-eluted fraction was adjusted to 7.0 by the addition of asmall volume of 10×PBS and stored at 4° C. for future analysis ifneeded.

[0202] The polypeptide elution fraction was concentrated to 5.0 ml usinga 5,000 molecular weight cutoff membrane concentrator (Millipore,Bedford, Mass.) according to the manufacturer's instructions. Toseparate z219aCEE polypeptide from free EE peptide, the concentratedpolypeptide elution fraction was subjected to chromatography on a 1.5×50cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) column equilibrated inPBS at a flow rate of 1.0 ml/min using a BioCad Sprint HPLC (PerSeptiveBioSystems, Framingham, Mass.). Two-ml fractions were collected and theabsorbance at 280 nM was monitored. The first peak of material absorbingat 280 nM and eluting near the void volume of the column was collected.This material represented purified z219aCEE polypeptide and was furthercharacterized by SDS-PAGE and Western blotting with anti-EE antibodies.

[0203] On Coomassie Blue stained SDS-PAGE gels, the z219aCEE polypeptidewas composed of three major bands of apparent molecular weights 26,000,80,000 and about 220,000. The mobility of these bands was the same onreducing and non-reducing gels. By western blotting on non-reducinggels, only the largest and the smallest molecular weight forms showedcross-reactivity with anti-EE antibodies. Under reducing conditions,only the 26,000 showed cross reactivity.

[0204] The protein concentration of the purified proteins was performedby BCA analysis (Pierce, Rockford, Ill.) and the material was aliquoted,and stored at −80° C. according to our standard procedures. Theconcentrations of z219aCEE polypeptide was 0.56 mg/ml.

[0205] To prepare anti-EE Sepharose, a 100 ml bed volume of proteinG-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with 100 mlof PBS containing 0.02% sodium azide using a 500 ml Nalgene 0.45 micronfilter unit. The gel was washed with 6.0 volumes of 200 mMtriethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an equalvolume of EE antibody solution containing 900 mg of antibody was added.After an overnight incubation at 4° C., unbound antibody was removed bywashing the resin with 5 volumes of 200 mM TEA as described above. Theresin was resuspended in 2 volumes of TEA, transferred to a suitablecontainer, and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.)dissolved in TEA, was added to a final concentration of 36 mg/ml ofprotein G-Sepharose gel. The gel was rocked at room temperature for 45min and the liquid was removed using the filter unit as described above.Nonspecific sites on the gel were then blocked by incubating for 10 min.at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA.The gel was then washed with 5 volumes of PBS containing 0.02% sodiumazide and stored in this solution at 4° C.

Example 8 Adenovirus Production of z219aCEE Polypeptide

[0206] Production of adenovirus containing z219aCEE was done accordingto the procedure of Becker et al., Meth. Cell Biol. 43:161-89, 1994.Briefly, the cDNA encoding z219aCEE was excised by EcoRI/XbaIrestriction digest from z219aCEE/pZP9 (Example 4). Restriction fragmentswere visualized by gel electrophoresis, excised and purified. Thez219aCEE restriction fragment was ligated into an EcoRI/XbaI digestedshuttle vector, pAC-CMV (Microbix Biosystems, Inc., Ontario, Canada).One microliter of the ligation reaction was electroporated into DH10Bcompetent cells (GIBCO BRL) according to manufacturer's direction andplated onto LB plates containing 50 μg/ml ampicillin, and incubatedovernight. Colonies were screened by restriction digest and large scaleplasmid DNA was prepared for positive clones.

[0207] The z219a containing shuttle vector, z219aCEE/pAC-CMV, wasco-transfected with El-deleted, adenovirus vector pJM17 (MicrobixBiosystems, Inc.) into 293 cells (ATCC CRL-1573) which express theadenovirus E1 gene. The co-transfection was done using a TransfectionMBS Mammalian Transfection Kit (Stratagene Cloning Systems, La Jolla,Calif.), according to the manufacturer's instructions. Virus propagationis conditional and is achieved only by growing the E1-deleted virus in acell line expressing the El gene. Recombinant virus is generated byhomologous recombination of overlapping fragments of the viral genome inthe pJM17 vector and the shuttle vector.

[0208] Cells were maintained for 2-4 weeks until the recombination eventoccurred. At that time, the host 293 cells were lysed by the virus,forming plaques of dead cells. Within 3-5 days the entire monolayer wascompletely lysed. The medium containing the viral lysate was collectedand any remaining intact cells were lysed by repeated freeze/thaw cyclesand the cell debris pelleted by centrifugation.

[0209] The viral lysate was then plaque purified according to the methodof Becker et al., ibid. Briefly, serial dilutions were prepared in DMEMcontaining 10% fetal bovine serum and 100 U/ml penicillin/streptomycin,plated on to monolayers of 293 cells and incubated at 37° C. for onehour. A melted 1.3% agarose/water solution was mixed with 2×DMEM(containing 4% FBS, 200 U/ml penicillin/streptomycin, 0.5 mg/mlfungizone and 30 mg/ml phenol red) and 6 ml was added to the virusinfected 293 cells followed by incubation at 37° C. until plaquesformed, 7-10 days. Single plaques were isolated and the presence of thez219aCEE insert was verified by PCR. Oligonucleotide primers wereZC12,700 (SEQ ID NO:25) and ZC12,742 (SEQ ID NO:26). Amplification wascarried out over 30 cycles of 94° C., 1 minute; 55° C., 1 minute 30seconds; and 72° C., 2 minutes; followed by a 10 minute extension at 72°C. The expected size of the PCR generated fragment was 1296. Theidentity of the insert was verified by sequence analysis.

[0210] One plaque from each construct was used to do a primaryamplification according to the methods of Becker et al., ibid . Briefly,30 dishes (150×25 mm) containing 293 cells at 80% confluence wereinfected at a multiplicity of infection of at least 10 pfu/cell. Cellswere incubated at 37° C. for 36-48 hours to allow for total lysis. Thelysate was harvested and 0.5% Nonidet P-40 was added followed by shakingat room temperature for 10 minutes to insure complete mixing. Celldebris was removed by centrifugation and the supernatant was incubated,with shaking, overnight in an 0.5 volume of 20% polyethyleneglycol/8000/2.5 M NaCl. The adenovirus was pelleted and resuspended in3-6 ml phosphate-buffered saline (PBS) and centrifuged to remove debris.Cesium chloride was added to the supernatant until 1 ml of solutionweighed 1.32-1.34 g. The solution was then subjected to high speedcentrifugation for 3 hours at 361,000 g. The white, adenovirus band wasrecovered. The virus solution was purified over a Pharmacia PD-10Sephadex column equilibrated with sterile PBS. The absorption ofcollected fractions was measured at 260 nm and peak fractions werepooled. The final concentration ranged from 1×10¹² to 1×10¹³ virions/mlas measured by optical density at 260 nm. A viral disruption assay wasdone to measure cytopathic effect by titration of virus on 293 cells tolook for cell lysis and measure infectivity of virus preps. For in vivouse of the virus, a second plaque purification was performed asdescribed above to measure plaque forming units.

[0211] Example 9

Z219a Transgenic Mice

[0212] Oligonucleotides were designed to generate a PCR fragmentcontaining a consensus Kozak sequence and the exact z219a coding region.These oligonucleotides were designed with an FseI site at the 5′ end andan AscI site at the 3′ end to facilitate cloning into pMT12-8, ourstandard transgenic vector. PMT12-8 contains the mouse MT-1 promoter anda 5′ rat insulin II intron upstream of the FseI site.

[0213] PCR reactions were carried out with 200 ng p219a template andoligonucleotides ZC 17184 (SEQ ID NO: 27) and ZC17185 (SEQ ID NO:28).PCR reaction conditions were as follows: 91° C. for 5 minutes followedby 54° C. before addition of KlenTaq™ polymerase (Clontech); 15 cyclesof 95° C. for 60 seconds, 62° C. for 60 seconds, and 72° C. for 90seconds; and 72° C. for 7 minutes. PCR products were separated byagarose gel electrophoresis and purified using a QiaQuick™ (Qiagen) gelextraction kit. The isolated, 746 bp, DNA fragment was digested withFseI and AscI (Boerhinger-Mannheim), ethanol precipitated and ligatedinto pMT12-8 that was previously digested with FseI and AscI. ThepMT12-8 plasmid, designed for expression of a gene of interest intransgenic mice, contains an expression cassette flanked by 10 kb ofMT-1 5′ DNA and 7 kb of MT-1 3′ DNA. The expression cassette comprisesthe MT-1 promoter, the rat insulin II intron, a polylinker for theinsertion of the desired clone, and the human growth hormone poly Asequence.

[0214] About one microliter of the ligation reaction was electroporatedinto DH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and grown in LB media containing 100 μg/ml ampicillin. MiniprepDNA was prepared from the picked clones and screened for the z219ainsert by restriction digestion with FseI and AscI, and subsequentagarose gel electrophoresis. Maxipreps of the correct pMT-z219aconstruct were performed. A SalI fragment containing with 5′ and 3′flanking sequences, the MT-1 promoter, the rat insulin II intron, z219acDNA and the human growth hormone poly A sequence was prepared and usedfor microinjection into fertilized murine oocytes.

[0215] Three transgenic mice were identified among 37 pups. All threegave little or no expression based on solution hybridization on liverRNA. This low percentage of transgenic mice suggests that highexpression of z219a is lethal in embryogenesis.

[0216] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 28 <210> SEQ ID NO 1<211> LENGTH: 876 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (119)...(823) <400>SEQUENCE: 1 gcccgcccct gcccgacacg accgctgccc gccccttgcc ttcctgacccaggggctccg 60 ctggctgcgg tcgcctggga gctgccgcca gggccaggag gggagcggcacctggaag 118 atg cgc cca ttg gct ggt ggc ctg ctc aag gtg gtg ttc gtg gtcttc 166 Met Arg Pro Leu Ala Gly Gly Leu Leu Lys Val Val Phe Val Val Phe1 5 10 15 gcc tcc ttg tgt gcc tgg tat tcg ggg tac ctg ctc gca gag ctcatt 214 Ala Ser Leu Cys Ala Trp Tyr Ser Gly Tyr Leu Leu Ala Glu Leu Ile20 25 30 cca gat gca ccc ctg tcc agt gct gcc tat agc atc cgc agc atc ggg262 Pro Asp Ala Pro Leu Ser Ser Ala Ala Tyr Ser Ile Arg Ser Ile Gly 3540 45 gag agg cct gtc ctc aaa gct cca gtc ccc aaa agg caa aaa tgt gac310 Glu Arg Pro Val Leu Lys Ala Pro Val Pro Lys Arg Gln Lys Cys Asp 5055 60 cac tgg act ccc tgc cca tct gac acc tat gcc tac agg tta ctc agc358 His Trp Thr Pro Cys Pro Ser Asp Thr Tyr Ala Tyr Arg Leu Leu Ser 6570 75 80 gga ggt ggc aga agc aag tac gcc aaa atc tgc ttt gag gat aac cta406 Gly Gly Gly Arg Ser Lys Tyr Ala Lys Ile Cys Phe Glu Asp Asn Leu 8590 95 ctt atg gga gaa cag ctg gga aat gtt gcc aga gga ata aac att gcc454 Leu Met Gly Glu Gln Leu Gly Asn Val Ala Arg Gly Ile Asn Ile Ala 100105 110 att gtc aac tat gta act ggg aat gtg aca gca aca cga tgt ttt gat502 Ile Val Asn Tyr Val Thr Gly Asn Val Thr Ala Thr Arg Cys Phe Asp 115120 125 atg tat gaa ggc gat aac tct gga ccg atg aca aag ttt att cag agt550 Met Tyr Glu Gly Asp Asn Ser Gly Pro Met Thr Lys Phe Ile Gln Ser 130135 140 gct gct cca aaa tcc ctg ctc ttc atg gtg acc tat gac gac gga agc598 Ala Ala Pro Lys Ser Leu Leu Phe Met Val Thr Tyr Asp Asp Gly Ser 145150 155 160 aca aga ctg aat aac gat gcc aag aat gcc ata gaa gca ctt ggaagt 646 Thr Arg Leu Asn Asn Asp Ala Lys Asn Ala Ile Glu Ala Leu Gly Ser165 170 175 aaa gaa atc agg aac atg aaa ttc agg tct agc tgg gta ttt attgca 694 Lys Glu Ile Arg Asn Met Lys Phe Arg Ser Ser Trp Val Phe Ile Ala180 185 190 gca aaa ggc ttg gaa ctc cct tcc gaa att cag aga gaa aag atcaac 742 Ala Lys Gly Leu Glu Leu Pro Ser Glu Ile Gln Arg Glu Lys Ile Asn195 200 205 cac tct gat gct aag aac aac aga tat tct ggc tgg cct gca gagatc 790 His Ser Asp Ala Lys Asn Asn Arg Tyr Ser Gly Trp Pro Ala Glu Ile210 215 220 cag ata gaa ggc tgc ata ccc aaa gaa cga agc tgacactgcagggtcctgag 843 Gln Ile Glu Gly Cys Ile Pro Lys Glu Arg Ser 225 230 235taaatgtgtt ctgtataaac aaatgcagct gga 876 <210> SEQ ID NO 2 <211> LENGTH:235 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 MetArg Pro Leu Ala Gly Gly Leu Leu Lys Val Val Phe Val Val Phe 1 5 10 15Ala Ser Leu Cys Ala Trp Tyr Ser Gly Tyr Leu Leu Ala Glu Leu Ile 20 25 30Pro Asp Ala Pro Leu Ser Ser Ala Ala Tyr Ser Ile Arg Ser Ile Gly 35 40 45Glu Arg Pro Val Leu Lys Ala Pro Val Pro Lys Arg Gln Lys Cys Asp 50 55 60His Trp Thr Pro Cys Pro Ser Asp Thr Tyr Ala Tyr Arg Leu Leu Ser 65 70 7580 Gly Gly Gly Arg Ser Lys Tyr Ala Lys Ile Cys Phe Glu Asp Asn Leu 85 9095 Leu Met Gly Glu Gln Leu Gly Asn Val Ala Arg Gly Ile Asn Ile Ala 100105 110 Ile Val Asn Tyr Val Thr Gly Asn Val Thr Ala Thr Arg Cys Phe Asp115 120 125 Met Tyr Glu Gly Asp Asn Ser Gly Pro Met Thr Lys Phe Ile GlnSer 130 135 140 Ala Ala Pro Lys Ser Leu Leu Phe Met Val Thr Tyr Asp AspGly Ser 145 150 155 160 Thr Arg Leu Asn Asn Asp Ala Lys Asn Ala Ile GluAla Leu Gly Ser 165 170 175 Lys Glu Ile Arg Asn Met Lys Phe Arg Ser SerTrp Val Phe Ile Ala 180 185 190 Ala Lys Gly Leu Glu Leu Pro Ser Glu IleGln Arg Glu Lys Ile Asn 195 200 205 His Ser Asp Ala Lys Asn Asn Arg TyrSer Gly Trp Pro Ala Glu Ile 210 215 220 Gln Ile Glu Gly Cys Ile Pro LysGlu Arg Ser 225 230 235 <210> SEQ ID NO 3 <211> LENGTH: 3 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Motif 1, corresponding to residues 127 to 129 of SEQ IDNO:2 <400> SEQUENCE: 3 Phe Asp Met 1 <210> SEQ ID NO 4 <211> LENGTH: 3<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Motif 2, corresponding to residues 156 to 158 of SEQID NO:2 <400> SEQUENCE: 4 Tyr Asp Asp 1 <210> SEQ ID NO 5 <211> LENGTH:3 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Motif 3, corresponding to residues 174 to 176of SEQ ID NO:2 <400> SEQUENCE: 5 Leu Gly Ser 1 <210> SEQ ID NO 6 <211>LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Motif 4, corresponding to residues 188to 190 of SEQ ID NO:2 <400> SEQUENCE: 6 Trp Val Phe 1 <210> SEQ ID NO 7<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Motif 5, corresponding toresidues 227 to 229 of SEQ ID NO:2 <400> SEQUENCE: 7 Glu Gly Cys 1 <210>SEQ ID NO 8 <211> LENGTH: 705 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: z219a Degeneratepolynucleotide sequence <220> FEATURE: <221> NAME/KEY: variation <222>LOCATION: (1)...(705) <223> OTHER INFORMATION: N is any nucleotide <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(705) <223>OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 8 atgmgnccnytngcnggngg nytnytnaar gtngtnttyg tngtnttygc nwsnytntgy 60 gcntggtaywsnggntayyt nytngcngar ytnathccng aygcnccnyt nwsnwsngcn 120 gcntaywsnathmgnwsnat hggngarmgn ccngtnytna argcnccngt nccnaarmgn 180 caraartgygaycaytggac nccntgyccn wsngayacnt aygcntaymg nytnytnwsn 240 ggnggnggnmgnwsnaarta ygcnaarath tgyttygarg ayaayytnyt natgggngar 300 carytnggnaaygtngcnmg nggnathaay athgcnathg tnaaytaygt nacnggnaay 360 gtnacngcnacnmgntgytt ygayatgtay garggngaya aywsnggncc natgacnaar 420 ttyathcarwsngcngcncc naarwsnytn ytnttyatgg tnacntayga ygayggnwsn 480 acnmgnytnaayaaygaygc naaraaygcn athgargcny tnggnwsnaa rgarathmgn 540 aayatgaarttymgnwsnws ntgggtntty athgcngcna arggnytnga rytnccnwsn 600 garathcarmgngaraarat haaycaywsn gaygcnaara ayaaymgnta ywsnggntgg 660 ccngcngarathcarathga rggntgyath ccnaargarm gnwsn 705 <210> SEQ ID NO 9 <211>LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC695 <400>SEQUENCE: 9 gatttaggtg acactatag 19 <210> SEQ ID NO 10 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer ZC7231 <400> SEQUENCE: 10tttttttttt tttttttttt tttttv 26 <210> SEQ ID NO 11 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer ZC13695 <400> SEQUENCE: 11cccttccgaa attcagagag 20 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC13789 <400> SEQUENCE: 12tccctgccca tctgacacct 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC13790 <400> SEQUENCE: 13ccagctgttc tcccataagt 20 <210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC14069 <400> SEQUENCE: 14cttggcatcg ttattcagtc t 21 <210> SEQ ID NO 15 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC13072 <400> SEQUENCE: 15aggtcctggg caagtgctgc 20 <210> SEQ ID NO 16 <211> LENGTH: 18 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC13956 <400> SEQUENCE: 16gtggtgttcg tggtcttc 18 <210> SEQ ID NO 17 <211> LENGTH: 18 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC13957 <400> SEQUENCE: 17cgatgctgcg gatgctat 18 <210> SEQ ID NO 18 <211> LENGTH: 243 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: z219aCEE polypeptide with C-terminal GluGlu tag <400>SEQUENCE: 18 Met Arg Pro Leu Ala Gly Gly Leu Leu Lys Val Val Phe Val ValPhe 1 5 10 15 Ala Ser Leu Cys Ala Trp Tyr Ser Gly Tyr Leu Leu Ala GluLeu Ile 20 25 30 Pro Asp Ala Pro Leu Ser Ser Ala Ala Tyr Ser Ile Arg SerIle Gly 35 40 45 Glu Arg Pro Val Leu Lys Ala Pro Val Pro Lys Arg Gln LysCys Asp 50 55 60 His Trp Thr Pro Cys Pro Ser Asp Thr Tyr Ala Tyr Arg LeuLeu Ser 65 70 75 80 Gly Gly Gly Arg Ser Lys Tyr Ala Lys Ile Cys Phe GluAsp Asn Leu 85 90 95 Leu Met Gly Glu Gln Leu Gly Asn Val Ala Arg Gly IleAsn Ile Ala 100 105 110 Ile Val Asn Tyr Val Thr Gly Asn Val Thr Ala ThrArg Cys Phe Asp 115 120 125 Met Tyr Glu Gly Asp Asn Ser Gly Pro Met ThrLys Phe Ile Gln Ser 130 135 140 Ala Ala Pro Lys Ser Leu Leu Phe Met ValThr Tyr Asp Asp Gly Ser 145 150 155 160 Thr Arg Leu Asn Asn Asp Ala LysAsn Ala Ile Glu Ala Leu Gly Ser 165 170 175 Lys Glu Ile Arg Asn Met LysPhe Arg Ser Ser Trp Val Phe Ile Ala 180 185 190 Ala Lys Gly Leu Glu LeuPro Ser Glu Ile Gln Arg Glu Lys Ile Asn 195 200 205 His Ser Asp Ala LysAsn Asn Arg Tyr Ser Gly Trp Pro Ala Glu Ile 210 215 220 Gln Ile Glu GlyCys Ile Pro Lys Glu Arg Ser Gly Ser Glu Tyr Met 225 230 235 240 Pro MetGlu <210> SEQ ID NO 19 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC14870 <400> SEQUENCE: 19 atcgtaggaa ttcatgcgcccattg 25 <210> SEQ ID NO 20 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC15101 <400> SEQUENCE: 20 tacgatggat ccgcttcgttcttt 24 <210> SEQ ID NO 21 <211> LENGTH: 8 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Glu-Glu tag sequence with linker <400> SEQUENCE: 21 Gly Ser Glu Tyr MetPro Met lu 1 5 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC13006 <400> SEQUENCE: 22 ggctgtcctc taagcgtcac20 <210> SEQ ID NO 23 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC13007 <400> SEQUENCE: 23 aggggtcaca gggatgcca19 <210> SEQ ID NO 24 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: EE peptidesequence <400> SEQUENCE: 24 Glu Tyr Met Pro Val Asp 1 5 <210> SEQ ID NO25 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC12700EQUENCE:25 ggaggtctat ataagcagag c 21 <210> SEQ ID NO 26 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC12742 EQUENCE:26ttatgtttca ggttcagggg 20 <210> SEQ ID NO 27 <211> LENGTH: 32 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC17184 EQUENCE:27 cgtacgggcgcgcctcagct tcgttctttg gg 32 <210> SEQ ID NO 28 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC17185 <400> SEQUENCE: 28cattcaggcc ggccaccatg cgcccattgg ct 32

What is claimed is:
 1. An isolated polynucleotide that encodes apolypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 26 (Tyr), to amino acid number 235 (Ser); and (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met), to amino acid number 235 (Ser).
 2. An isolated polynucleotidemolecule selected from the group consisting of: (a) polynucleotidemolecules comprising a nucleotide sequence as shown in SEQ ID NO:1 fromnucleotide 194 to nucleotide 823; (b) polynucleotide moleculescomprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide119 to nucleotide 823; and (c) polynucleotide molecules complementary to(a) or (b).
 3. An isolated polynucleotide sequence according to claim 1,wherein the polynucleotide comprises nucleotide 1 to nucleotide 705 ofSEQ ID NO:8.
 4. An isolated polynucleotide according to claim 1, whereinthe polypeptide consists of a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence as shown in SEQ ID NO:2from amino acid number 26 (Tyr), to amino acid number 235 (Ser).
 5. Anisolated polynucleotide according to claim 4, wherein the z219apolypeptide consists of a sequence of amino acid residues as shown inSEQ ID NO:2 from amino acid number 26 (Tyr), to amino acid number 235(Ser).
 6. The isolated polynucleotide molecule of claim 1, wherein thepolynucleotide encodes a polypeptide that contains motifs 1, 2, 3, 4 and5 spaced apart from N-terminus to C-terminus in a configurationM1-{25-26}-M2-{15}-M3-{11}-M4-{34-36}-M5.
 7. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a z219a polypeptide that is at least90% identical to an amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 26 (Tyr), to amino acid number 235 (Ser); and atranscription terminator, wherein the promoter is operably linked to theDNA segment, and the DNA segment is operably linked to the transcriptionterminator.
 8. An expression vector according to claim 7, furthercomprising a secretory signal sequence operably linked to the DNAsegment.
 9. A cultured cell into which has been introduced an expressionvector according to claim 7, wherein the cell expresses the polypeptideencoded by the DNA segment.
 10. A DNA construct encoding a fusionprotein, the DNA construct comprising: a first DNA segment encoding apolypeptide that is at least 90% identical to a sequence of amino acidresidues 1 (Met) through 25 (Gly) of SEQ ID NO:2; and second DNA segmentencoding an additional polypeptide, wherein the first and second DNAsegments are connected in-frame; and encode the fusion protein.
 11. Anisolated polypeptide comprising a sequence of amino acid residues thatis at least 90% identical to an amino acid sequence selected from thegroup consisting of: (a) polypeptide molecules comprising an amino acidsequence as shown in SEQ ID NO:2 from amino acid number 26 (Tyr) toamino acid number 235 (Ser) of SEQ ID NO:2; and (b) polypeptidemolecules comprising an amino acid sequence as shown in SEQ ID NO:2 fromamino acid residue number 1 (Met) to amino acid residue number 235(Ser).
 12. An isolated polypeptide according to claim 11, wherein thepolypeptide consists of a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence as shown in SEQ ID NO:2from amino acid number 26 (Tyr) to amino acid number 235 (Ser).
 13. Anisolated polypeptide according to claim 12, wherein the sequence ofamino acid residues is as shown in SEQ ID NO:2 from amino acid number 26(Tyr) to amino acid number 235 (Ser).
 14. The isolated polypeptide ofclaim 11, wherein the polypeptide molecule encodes motifs 1, 2, 3, 4 and5 spaced apart from N-terminus to C-terminus in a configurationM1-{25-26}-M2-{15}-M3-{11}-M4-{34-36}-M5.
 15. A method of producing az219a polypeptide comprising: culturing a cell according to claim 9; andisolating the z219a polypeptide produced by the cell.
 16. A method ofproducing an antibody to z219a polypeptide comprising: inoculating ananimal with a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 9 to 210 amino acids, wherein the polypeptideis at least 90% identical to a contiguous sequence of amino acids in SEQID NO:2 from amino acid number 26 (Tyr) to amino acid number 235 (Ser);(b) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2from amino acid number 26 (Tyr) to amino acid number 235 (Ser); (c) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 59 (Arg) to amino acid number 133 (Asp); (d) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 135 (Ser) to amino acid number 212 (Ala); (e) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid 215 (Asn) to amino acid number 231 (Pro); and wherein thepolypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal.
 17. An antibodyproduced by the method of claim 16, which specifically binds to a z219apolypeptide.
 18. The antibody of claim 17, wherein the antibody is amonoclonal antibody.
 19. An antibody which specifically binds to apolypeptide of claim
 11. 20. A method of detecting, in a test sample,the presence of an antagonist of z219a protein activity, comprising:transfecting a z219a-responsive cell, with a reporter gene constructthat is responsive to a z219a-stimulated cellular pathway; and producinga z219a polypeptide by the method of claim 15; and adding the z219apolypeptide to the cell, in the presence and absence of a test sample;and comparing levels of response to the z219a polypeptide, in thepresence and absence of the test sample, by a biological or biochemicalassay; and determining from the comparison, the presence of theantagonist of z219a activity in the test sample.
 21. A method ofdetecting, in a test sample, the presence of an agonist of z219a proteinactivity, comprising: transfecting a z219a-responsive cell, with areporter gene construct that is responsive to a z219a-stimulatedcellular pathway; and adding a test sample; and comparing levels ofresponse in the presence and absence of the test sample, by a biologicalor biochemical assay; and determining from the comparison, the presenceof the agonist of z219a activity in the test sample.